Peptide nanoparticles and uses therefor

ABSTRACT

The present invention provides nanoparticle compositions including one or more peptides. The present invention achieves transdermal delivery of such peptides without the need for peptide modification, or for use of chemical or mechanical abrasion or disruption of skin.

RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119(e) to U.S.provisional patent application, U.S. Ser. No. 60/872,206, filed Dec. 1,2006 (“the '206 application”). The entire contents of the '206application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Peptides have been shown to have beneficial cosmetic and therapeuticeffects on the skin. In experimental models, short peptides (of lengthup to 30 amino acids) have been shown to stimulate collagen growth inthe extra-cellular matrix of the skin, which may improve the appearanceof skin as well as improve the healing of damaged skin (Katayama, etal., 1993, J. Biol. Chem., 268:9941; incorporated herein by reference).Modified peptides have also been shown to decrease the appearance ofwrinkles through the modulation of enzymes that influence muscularcontractions in the muscles underlying the skin that contribute towrinkle formation (Lupo, 2005, Dermatol. Surg., 31:832; incorporatedherein by reference).

However, a major problem in achieving the potential cosmetic andtherapeutic effects of these peptides in humans has been the transdermaldelivery of the peptides across the outer skin barrier (stratum corneum)to the site of biological action, e.g., the extra-cellular matrix orunderlying muscle (Robinson, et al., 2005, International J. CosmeticScience 27:155; incorporated herein by reference). To achieve thedelivery of transdermal delivery of the peptides in humans, the peptideshave had to be chemically modified by the addition of chemical moietiessuch as but not limited to acetyl and/or palmitoyl groups (Robinson, etal., supra). These chemical modifications are disadvantageous becausethey are expensive and time-consuming, which negatively impact thecommercial manufacture of a product containing these peptides. Chemicalmodifications of the peptides can also decrease the biological activityof the peptide by decreasing its ability to bind at the cellularreceptor site of biological activity (through, for example, stericinterference), thus making it less effective. A peptide that is lesseffective biologically would be less effective for cosmetic ortherapeutic purposes. Comparably, a peptide that is less effectivebiologically would need to be administered at higher levels to achieveits desired biological effect (if the effect were even possible), whichwould be a cost disadvantage for the commercial manufacture of aproduct.

SUMMARY OF THE INVENTION

The present invention describes nanoparticles that incorporateunmodified short peptides (2 to 30 amino acids long) that arebiologically active agents in the skin (including epidermis and dermis),sub-cutaneous tissue (including adipose tissue), and contiguous muscles.

Inventive nanoparticles can be applied to the skin of a subject. In someembodiments, inventive nanoparticles achieve transdermal delivery ofincorporated peptides to the subject.

Inventive nanoparticles can be applied to the skin as a simplesuspension or dispersion or mixed with one or more excipients andprepared as a formulation such as, but not limited to, a skin softener,nutrition lotion, cleansing lotion, cleansing cream, skin milk,emollient lotion, massage cream, emollient cream, make-up base,lipstick, facial pack or facial gel, cleaner formulation (e.g. shampoos,rinses, body cleanser, hair-tonics, and soaps), and dermatologicalcomposition (e.g. lotions, ointments, gels, creams, patches and sprays).

Thus, the present invention provides systems and compositions for thetransdermal delivery of unmodified peptides. Among the many advantagesof this invention is the ability to delivery peptides without injectionand further without a requirement for mechanical or chemical abrasion oralteration of skin. Additional advantages include an ability to utilizeunmodified peptides, thereby simplifying and reducing the cost ofproduction of inventive cosmetic and/or pharmaceutical preparations and,further, preserving biological activity of the peptide.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. Histological Analysis of Mice Treated with PeptideNanoparticles. Shown are photomicrographs of skin tissue stained withMasson's Trichrome stain. The average histologic score was 2.33 out of apossible 4 in the Control Group (nanoparticle formulation withoutpentapeptide). The average histologic score was 3.67 out of a possible 4in the Treatment Group (nanoparticle formulation with pentapeptide).

DEFINITIONS

Abrasion: The term “abrasion,” as used herein refers to any means ofaltering, disrupting, removing, or destroying the top layer of the skin.In some embodiments, abrasion refers to a mechanical means of altering,disrupting, removing, or destroying the top layer of the skin. In someembodiments, abrasion refers to a chemical means of altering,disrupting, removing, or destroying the top layer of skin. To give but afew examples, agents such as exfoliants, fine particles (e.g. magnesiumor aluminum particles), acids (e.g. alpha-hydroxy acids or beta-hydroxyacids), alcohols, may cause abrasion. In general, permeation enhancerssuch as those described, for example, by Donovan (e.g., U.S. PatentPublications 2004/009180 and 2005/175636 and PCT Publication WO04/06954; all of which are incorporated herein by reference), and Graham(e.g., U.S. Pat. No. 6,939,852 and U.S. Patent Publication 2006/093624;both of which are incorporated herein by reference), etc., are expectedto cause abrasion. Of course, those of ordinary skill in the art willappreciate that a particular agent may cause abrasion when present atone concentration, or in association with one or more other agents, butmay not cause abrasion under different circumstances. Thus, whether ornot a particular material is an “abrasive agent” depends on context.Abrasion can readily be assessed by those of ordinary skill in the art,for example by observation of redness or irritation of the skin and/orhistologic examination of skin showing alteration, disruption, removal,or erosion of the stratum corneum.

Amino acid: As used herein, term “amino acid,” in its broadest sense,refers to any compound and/or substance that can be incorporated into apolypeptide chain. In some embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In some embodiments, an amino acid is anaturally-occurring amino acid. In some embodiments, an amino acid is asynthetic amino acid; in some embodiments, an amino acid is a D-aminoacid; in some embodiments, an amino acid is an L-amino acid. “Standardamino acid” refers to any of the twenty standard L-amino acids commonlyfound in naturally occurring peptides. “Nonstandard amino acid” refersto any amino acid, other than the standard amino acids, regardless ofwhether it is prepared synthetically or obtained from a natural source.Amino acids, including carboxy- and/or amino-terminal amino acids inpeptides, can be modified by methylation, amidation, acetylation, and/orsubstitution with other chemical groups that can change the peptide'scirculating half-life without adversely affecting their activity.However, as described herein, the present invention is specificallydirected to “unmodified peptides”, meaning peptides that have not beenchemically modified in order to facilitate or achieve transdermaldelivery. Amino acids may participate in a disulfide bond. The term“amino acid” is used interchangeably with “amino acid residue,” and mayrefer to a free amino acid and/or to an amino acid residue of a peptide.It will be apparent from the context in which the term is used whetherit refers to a free amino acid or a residue of a peptide.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, and/or worms. In some embodiments, ananimal may be a transgenic animal, genetically-engineered animal, and/ora clone.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value).

Biologically active agent: As used herein, the phrase “biologicallyactive agent” refers to any substance that has activity in a biologicalsystem and/or organism. For instance, a substance that, whenadministered to an organism, has a biological effect on that organism,is considered to be biologically active. In particular embodiments,where a protein or polypeptide is biologically active, a portion of thatprotein or polypeptide that shares at least one biological activity ofthe protein or polypeptide is typically referred to as a “biologicallyactive” portion.

Botulinum toxin: The term “botulinum toxin,” as used herein, refers toany neurotoxin produced by Clostridium botulinum. Except as otherwiseindicated, the term encompasses fragments or portions (e.g., the lightchain and/or the heavy chain) of such neurotoxin that retain appropriateactivity (e.g., muscle relaxant activity). The phrase “botulinum toxin,”as used herein, encompasses the botulinum toxin serotypes A, B, C, D, E,F, and G. Botulinum toxin, as used herein, also encompasses both abotulinum toxin complex (i.e., for example, the 300, 600, and 900 kDcomplexes) as well as the purified (i.e., for example, isolated)botulinum toxin (i.e., for example, about 150 kD). “Purified botulinumtoxin” is defined as a botulinum toxin that is isolated, orsubstantially isolated, from other proteins, including protein that fora botulinum toxin complex. A purified toxin may be greater than 95%pure, and preferably is greater than 99% pure. Those of ordinary skillin the art will appreciate that the present invention is not limited toany particular source of botulinum toxin. For example, botulinum toxinfor use in accordance with the present invention may be isolated fromClostridium botulinum, may be chemically synthesized, may be producedrecombinantly (i.e., in a host cell or organism other than Clostridiumbotulinum), etc.

Characteristic portion: As used herein, the phrase a “characteristicportion” of a substance, in the broadest sense, is one that shares somedegree of sequence and/or structural identity and/or at least onefunctional characteristic with the relevant intact substance. Forexample, a “characteristic portion” of a protein or polypeptide is onethat contains a continuous stretch of amino acids, or a collection ofcontinuous stretches of amino acids, that together are characteristic ofa protein or polypeptide. In some embodiments, each such continuousstretch generally will contain at least 2, 5, 10, 15, 20 or more aminoacids. In general, a characteristic portion is one that, in addition tothe sequence identity specified above, shares at least one functionalcharacteristic with the relevant intact protein. In some embodiments,the characteristic portion may be biologically active.

Hydrophilic: As used herein, a “hydrophilic” substance is a substancethat may be soluble in polar solvents. In some embodiments, ahydrophilic substance can transiently bond with polar solvents. In someembodiments, a hydrophilic substance transiently bonds with polarsolvents through hydrogen bonding. In some embodiments, the polarsolvent is water. In some embodiments, a hydrophilic substance may beionic. In some embodiments, a hydrophilic substance may be non-ionic. Insome embodiments, a hydrophilic substance may dissolve more readily inwater, polar solvents, or hydrophilic solvents than in oil, non-polarsolvents, or hydrophobic solvents. In some embodiments, a hydrophilicsubstance may dissolve less readily in oil, non-polar solvents, orhydrophobic solvents than in water, polar solvents, or hydrophilicsolvents. In some embodiments, a substance is hydrophilic relative toanother substance because it is more soluble in water, polar solvents,or hydrophilic solvents than is the other substance. In someembodiments, a substance is hydrophilic relative to another substancebecause it is less soluble in oil, non-polar solvents, or hydrophobicsolvents than is the other substance.

Hydrophobic: As used herein, a “hydrophobic” substance is a substancethat may be soluble in non-polar solvents. In some embodiments, ahydrophobic substance is repelled from polar solvents. In someembodiments, the polar solvent is water. In some embodiments,hydrophobic substances are non-polar. In some embodiments, a hydrophobicsubstance may dissolve more readily in oil, non-polar solvents, orhydrophobic solvents than in water, polar solvents, or hydrophilicsolvents. In some embodiments, a hydrophobic substance may dissolve lessreadily in water, polar solvents, or hydrophilic solvents than in oil,non-polar solvents, or hydrophobic solvents. In some embodiments, asubstance is hydrophobic relative to another substance because it ismore soluble in oil, non-polar solvents, or hydrophobic solvents than isthe other substance. In some embodiments, a substance is hydrophobicrelative to another substance because it is less soluble in water, polarsolvents, or hydrophilic solvents than is the other substance.

In conjunction with: As used herein, the phrase “delivered inconjunction with” refers to the co-delivery of two or more substances oragents. In particular, according to the present invention, the phrase isused herein in reference to delivery of a biologically active agent withinventive nanoparticles and/or nanoparticle compositions. A substance oragent is delivered in conjunction with nanoparticles when the substanceor agent is combined with nanoparticles and/or nanoparticlecompositions; is encapsulated or completely surrounded by nanoparticles;is embedded within an nanoparticle micellar membrane; and/or isassociated with the outer surface of an nanoparticle micellar membrane.A substance or agent to be delivered in conjunction with nanoparticlesand/or nanoparticle compositions may or may not be covalently linked tothe nanoparticles and/or nanoparticle compositions. A substance or agentto be delivered in conjunction with inventive nanoparticles and/ornanoparticle compositions may or may not be attached to thenanoparticles and/or nanoparticle compositions by adsorption forces.

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, or more of the other components with which they were initiallyassociated. In some embodiments, isolated substances and/or entities aremore than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% pure.

Microfluidized: As used herein, the term “microfluidized” means exposedto high shear forces. In some embodiments, such exposure to high shearforces is accomplished by exposure to high pressure; in some embodimentssuch high pressure is within the range of about 15,000 to about 26,000psi. In some embodiments, such exposure to high shear forces isaccomplished by cavitation. In some embodiments, such exposure to highshear forces is accomplished by passing a sample through an instrumentsuch as, for example, a Microfluidizer® (Microfluidics Corporation/MFICCorporation) or other like device that may be useful in creating auniform nanoparticle composition. In some embodiments of the presentinvention, a sample is microfluidized through exposure to high shearforces for a period of time less than about 10 minutes. In someembodiments, the period of time is less than about 9, 8, 7, 6, 5, 4, 3,2, or 1 minute(s). In some embodiments, the period of time is within therange of about 1-2 minutes. In some embodiments, the period of time isabout 30 seconds. In some embodiments of the invention, a sample is“microfluidized” through a single exposure to high shear forces; suchembodiments are referred to as “single pass” microfluidization.

Nanoparticle: As used herein, the term “nanoparticle” refers to anyparticle having a diameter of less than 1000 nanometers (nm). In someembodiments, a nanoparticle has a diameter of less than 300 nm, asdefined by the National Science Foundation. In some embodiments, ananoparticle has a diameter of less than 100 nm as defined by theNational Institutes of Health. In some embodiments, nanoparticles aremicelles in that they comprise an enclosed compartment, separated fromthe bulk solution by a micellar membrane. A “micellar membrane”comprises amphiphilic entities which have aggregated to surround andenclose a space or compartment (e.g., to define a lumen).

Nanoparticle composition: As used herein, the term “nanoparticlecomposition” refers to any substance that contains at least onenanoparticle. In some embodiments, a nanoparticle composition is auniform collection of nanoparticles. In some embodiments, nanoparticlecompositions are dispersions or emulsions. In general, a dispersion oremulsion is formed when at least two immiscible materials are combined.An “oil-in-water” dispersion is one in which oily particles (orhydrophobic or non-polar) are dispersed within an aqueous dispersionmedium. A “water-in-oil” dispersion is one in which aqueous (orhydrophilic or polar) particles are dispersed within an oily dispersionmedium. Those of ordinary skill in the art will appreciate that adispersion can be formed from any two immiscible media and is notlimited strictly to combinations of aqueous and oily media. The term“dispersion medium” therefore applies broadly to any dispersion mediumnotwithstanding that it is common to refer to “aqueous” and “oily”categories. In some embodiments, nanoparticle compositions arenanoemulsions. In some embodiments, nanoparticle compositions comprisemicelles. In some particular embodiments, a nanoparticle compositioncomprises amphiphilic entity nanoparticles as described in PCTapplication serial number PCT/US07/______, entitled “AMPHIPHILIC ENTITYNANOPARTICLES,” filed on Nov. 30, 2007 (incorporated herein byreference). In some embodiments, a nanoparticle composition is stable.In some embodiments, a nanoparticle composition includes one or morebiologically active agents to be delivered in conjunction with thenanoparticles.

Nutraceutical: As used herein, the term “nutraceutical” refers to anysubstance thought to provide medical, health, or biological benefits. Insome embodiments, nutraceuticals may prevent disease. In someembodiments, nutraceuticals may provide basic nutritional value. In someembodiments, a nutraceutical is a food or part of a food. In someembodiments, a nutraceutical agent may be a class of isolated nutrients,dietary supplements, vitamins, minerals, herbs, fortified foods, healingfoods, genetically engineered foods, and processed foods. Nutraceuticalsmay also be known as “phytochemical foods” or “functional foods.”

Premix: As used herein, the term “premix” refers to any combination ofcomponents that is subsequently used to generate a nanoparticlecomposition according to the present invention. For example, a premix isany collection of ingredients that, when subjected to high shear forces,generates nanoparticles according to the present invention. In someembodiments, a premix contains two or more immiscible solvents. In someembodiments, a premix contains components that self-assemble intonanoparticles. In some embodiments, a premix contains components thatself-assemble into micelles. In some embodiments, a premix contains oneor more amphiphilic entities as described in PCT application serialnumber PCT/US07/______, entitled “AMPHIPHILIC ENTITY NANOPARTICLES,”filed Nov. 30, 2007 (incorporated herein by reference). In someembodiments, a premix contains one or more unmodified peptides; in someembodiments, a premix contains at least one other biologically activeagent. In some embodiments, a premix is agitated, mixed, and/or stirred;in some embodiments, a premix is agitated, mixed, and/or stirred priorto being subjected to high shear force. In some embodiments, a premixcomprises at least one solubilized component (i.e., at least onecomponent that is in solution); in some such embodiments, the premix issubjected to high shear force after such solubilization is achieved.

Pure: As used herein, a substance and/or entity is “pure” if it issubstantially free of other components. For example, a preparation thatcontains more than about 90% of a particular substance and/or entity istypically considered to be a pure preparation. In some embodiments, asubstance and/or entity is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99% pure.

Shear force: As used herein, the term “shear force” refers to a forcethat is parallel to the face of a material, as opposed to a force thatis perpendicular to the face of a material. In some embodiments, acomposition exposed to high shear forces in order to produce a uniformnanoparticle composition. Any method known in the art can be used togenerate high shear forces. In some embodiments, cavitation is used togenerate high shear forces. In some embodiments, high pressurehomogenization is used to generate high shear forces. Alternatively oradditionally, high shear force may be administered by exposure to highpressure, for example about 15,000 psi. In some embodiments, such highpressure is within the range of about 18,000 to about 26,000 psi; insome embodiments, it is within the range of about 20,000 to about 25,000psi. In some embodiments, a Microfluidizer® Processor (MicrofluidicsCorporation/MFIC Corporation) or other like device is used to generatehigh shear force. Microfluidizer® Processors provide high pressure and aresultant high shear rate by accelerating a composition throughmicrochannels (typically having dimensions on the order of 75 microns)at a high velocity (typically in the range of 50 m/s-300 m/s) for sizereduction to the nanoscale range. As the fluid exits the microchannelsit forms jets which collide with jets from opposing microchannels. Inthe channels the fluid experiences high shear (up to 10⁷ l/s) which isorders of magnitude higher than that of conventional technologies. Jetcollisions result in mixing in submicron level. Therefore, in suchdevices, high shear and/or impact can achieve particle size reductionand mixing of multiphase. In some embodiments of the present invention,a sample is exposed to high shear forces for a period of time less thanabout 10 minutes. In some embodiments, the period of time is less thanabout 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2,or about 1 minute(s). In some embodiments, the period of time is withinthe range of about 1 to about 2 minutes or less; in some embodiments,the period of time is about 30 seconds. In some embodiments of theinvention, a sample is “microfluidized” through a single exposure tohigh shear forces; such embodiments are referred to herein as “singlepass” microfluidization.

Small Molecule: In general, a “small molecule” is understood in the artto be an organic molecule that is less than about 5 kilodaltons (Kd) insize. In some embodiments, the small molecule is less than about 3 Kd,about 2 Kd, or about 1 Kd. In some embodiments, the small molecule isless than about 800 daltons (D), about 600 D, about 500 D, about 400 D,about 300 D, about 200 D, or about 100 D. In some embodiments, smallmolecules are non-polymeric. In some embodiments, small molecules arenot proteins, peptides, or amino acids. In some embodiments, smallmolecules are not nucleic acids or nucleotides. In some embodiments,small molecules arc not saccharides or polysaccharides.

Subject: As used herein, the term “subject” or “patient” refers to anyorganism to which a composition of this invention may be administered,e.g., for experimental, diagnostic, prophylactic, and/or therapeuticpurposes. Typical subjects include animals (e.g., mammals such as mice,rats, rabbits, non-human primates, and humans; insects; worms; etc.).

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Stable: The term “stable,” when applied to nanoparticle compositionsherein, means that the compositions maintain one or more aspects oftheir physical structure (e.g., size range and/or distribution ofparticles) over a period of time. In some embodiments of the invention,a stable nanoparticle composition is one for which the average particlesize, the maximum particle size, the range of particle sizes, and/or thedistribution of particle sizes (i.e., the percentage of particles abovea designated size and/or outside a designated range of sizes) ismaintained for a period of time. In some embodiments, the period of timeis at least about one hour; in some embodiments the period of time isabout 5 hours, about 10 hours, about one (1) day, about one (1) week,about two (2) weeks, about one (1) month, about two (2) months, aboutthree (3) months, about four (4) months, about five (5) months, aboutsix (6) months, about eight (8) months, about ten (10) months, abouttwelve (12) months, about twenty-four (24) months, or longer. In someembodiments, the period of time is within the range of about one (1) dayto about twenty-four (24) months, about two (2) weeks to about twelve(12) months, about two (2) months to about five (5) months, etc. Forexample, if a nanoparticle composition is subjected to prolongedstorage, temperature changes, and/or pH changes and a majority of thenanoparticles in the population maintain a diameter within a statedrange (i.e., for example, between approximately 10 nm-120 nm), thenanoparticle composition is stable. For some such populations, amajority is more than about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%,about 99.6%, about 99.7%, about 99.8%, about 99.9%, or more. In someembodiments of the invention, where a nanoparticle composition comprisesone or more biologically active agents (e.g. unmodified peptide), thenanoparticle composition is considered stable if the concentration ofbiologically active agent is maintained in the composition over thedesignated period of time under a designated set of conditions.

Substantially free of An inventive nanoparticle composition is said tobe “substantially free of” particles whose diameter is outside of astated range when no more than about 50% of the particles in thatcomposition have diameters outside of the range. In some embodiments, nomore than 25% of the particles are outside of the range. In someembodiments, no more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or less of the particleshave diameters outside of the stated range.

Suffering from: An individual who is “suffering from” a disease,disorder, or condition (e.g., facial wrinkles) has been diagnosed withor exhibits symptoms of the disease, disorder, or condition.

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” means an amount of inventivenanoparticle composition that is sufficient, when administered to apatient suffering from or susceptible to a disease, disorder, and/orcondition, to treat the disease, disorder, and/or condition.

Therapeutic agent: As used herein, the phrase “therapeutic agent” refersto any agent that, when administered to a subject, has a therapeuticeffect and/or elicits a desired biological and/or pharmacologicaleffect.

Treatment: As used herein, the term “treatment” (also “treat” or“treating”) refers to any administration of a biologically active agentthat partially or completely alleviates, ameliorates, relives, inhibits,delays onset of, reduces severity of and/or reduces incidence of one ormore symptoms or features of a particular disease, disorder, and/orcondition. Such treatment may be of a subject who does not exhibit signsof the relevant disease, disorder and/or condition and/or of a subjectwho exhibits only early signs of the disease, disorder, and/orcondition. Alternatively or additionally, such treatment may be of asubject who exhibits one or more established signs of the relevantdisease, disorder and/or condition.

Toxic solvent: As used herein, the term “toxic solvent” refers to anysubstance that may alter, disrupt, remove, or destroy an animal'stissue. As would be understood by one of ordinary skill in the art, ananimal's tissue can include living cells, dead cells, extracellularmatrix, cellular junctions, biological molecules, etc. To give but a fewexamples, toxic solvents include dimethyl sulfoxide, dimethyl acetimide,dimethyl foramide, chloroform, tetramethyl foramide, acetone, acetates,and alkanes.

Uniform: The term “uniform,” when used herein in reference to ananoparticle composition, refers to a nanoparticle composition in whichthe individual nanoparticles have a specified range of particle diametersizes. For example, in some embodiments, a uniform nanoparticlecomposition is one in which the difference between the minimum diameterand maximum diameter does not exceed approximately 600, approximately550, approximately 500, approximately 450, approximately 400,approximately 350, approximately 300, approximately 250, approximately200, approximately 150, approximately 100, approximately 90,approximately 80, approximately 70, approximately 60, approximately 50,or fewer nm. In some embodiments, particles (e.g.,unmodified-peptide-containing particles) within inventive uniformnanoparticle compositions have diameters that are smaller than about600, about 550, about 500, about 450, about 400, about 350, about 300,about 250, about 200, about 150, about 130, about 120, about 115, about110, about 100, about 90, about 80 nm, or less. In some embodiments,particles (e.g., unmodified-peptide-containing particles) withininventive uniform nanoparticle compositions have diameters within therange of about 10 and about 600 nanometers. In some embodiments,particles (e.g., unmodified-peptide-containing particles) withininventive uniform nanoparticle compositions have diameters within therange of about 10 to about 300, about 10 to about 200, about 10 to about150, about 10 to about 130, about 10 to about 120, about 10 to about115, about 10 to about 110, about 10 to about 100, or about 10 to about90 nm. In some embodiments, particles (e.g.,unmodified-peptide-containing particles) within inventive botulinumnanoparticle compositions have an average particle size that is underabout 300, about 250, about 200, about 150, about 130, about 120, about115, about 110, about 100, or about 90 nm. In some embodiments, theaverage particle size is within the range of about about 10 to about300, about 50 to about 250, about 60 to about 200, about 65 to about150, about 70 to about 130 nm. In some embodiments, the average particlesize is about 80 to about 110 nm. In some embodiments, the averageparticle size is about 90 to about 100 nm. In some embodiments, amajority of the particles (e.g., unmodified-peptide-containingparticles) within inventive uniform nanoparticle compositions havediameters below a specified size or within a specified range. In someembodiments, the majority is more than 50%, 60%, 70%, 75%, 80%, 85%,90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or moreof the particles in the composition. In some embodiments of theinvention, a uniform nanoparticle composition is achieved bymicrofluidization of a sample. In some embodiments of the invention, auniform nanoparticle composition is prepared by exposure to high shearforce, e.g., by microfluidization.

Unmodified peptide: As used herein, the term “unmodified peptide” refersto a peptide that has not been chemically modified through the additionof other covalently-bonded functional groups intended to achievetransdermal delivery of the peptide. In some embodiments, the peptidehas not been chemically modified to add pendant acetyl and/or palmitoylgroups. In some embodiments, the peptide has not been chemicallymodified to add any functional pendant groups.

DESCRIPTION OF CERTAIN EMBODIMENTS Nanoparticles

As discussed herein, the present invention provides nanoparticlecompositions that include one or more unmodified peptides. In someembodiments, such nanoparticle compositions further include one or moreother biologically active agents in addition to the unmodified peptides.In some embodiments, the nanoparticle compositions are formulated withone or more other components, for example in a pharmaceutical orcosmetic preparation. In some embodiments, such a pharmaceutical orcosmetic preparation is formulated to achieve transdermal delivery ofthe unmodified peptides (and/or one or more other biologically activeagents).

In some embodiments, inventive nanoparticle compositions are stable. Insome embodiments, the nanoparticle compositions are uniform.

In some embodiments, a uniform nanoparticle composition comprises apopulation of particles whose difference between the minimum and maximumdiameters does not exceed approximately 600 nm, approximately 550 nm,approximately 500 nm, approximately 450 nm, approximately 400 nm,approximately 350 nm, approximately 300 nm, approximately 250 nm,approximately 200 nm, approximately 150 nm, or approximately 100 nm.

In some embodiments, inventive nanoparticles have diameters that aresmaller than about 1000, about 600, about 550, about 500, about 450,about 400, about 350, about 300, about 250, about 200, about 150, about130, about 120, about 115, about 110, about 100, about 90, about 80,about 50 nm, or less.

In some embodiments, inventive nanoparticles have a diameter of 1 nm to1000 nm, 1 nm to 600 nm, 1 nm to 500 nm, 1 nm to 400 nm, 1 nm to 300 nm,1 nm to 200 nm, 1 nm to 150 nm, 1 nm to 120 nm, 1 nm to 100 nm, 1 nm to75 nm, 1 nm to 50 nm, or 1 nm to 25 nm. In some embodiments, inventivenanoparticles have a diameter of 1 nm to 15 nm, 15 nm to 200 nm, 25 nmto 200 nm, 50 nm to 200 nm, or 75 nm to 200 nm.

In some embodiments, the total particle distribution is encompassedwithin the specified range of particle diameter size. In someembodiments, less than 50%, 25%, 10%, 5%, or 1% of the total particledistribution is outside of the specified range of particle diametersizes. In some embodiments, less than 1% of the total particledistribution is outside of the specified range of particle diametersizes. In certain embodiments, the nanoparticle composition issubstantially free of particles having a diameter larger than 300 nm,250 nm, 200 nm, 150 nm, 120 nm, 100 nm, 75 nm, 50 nm, or 25 nm.

In some embodiments, nanoparticles within inventive nanoparticlecompositions have an average particle size that is under about 300 nm,about 250 nm, about 200 nm, about 150 nm, about 130 nm, about 120 nm,about 115 nm, about 110 nm, about 100 nm, about 90 nm, or about 50 nm.In some embodiments, the average particle size is within the range ofabout 10 nm to about 300 nm, 50 nm to about 250 nm, 60 nm to about 200nm, 65 nm to about 150 nm, or 70 nm to about 130 nm. In someembodiments, the average particle size is about 80 nm to about 110 nm.In some embodiments, the average particle size is about 90 to about 100nm.

In some embodiments, inventive nanoparticle compositions aresubstantially free of particles having a diameter in excess of 300 nm.Specifically, in some embodiments, fewer than 50%, of the nanoparticlesin inventive nanoparticle compositions have a diameter in excess of 300nm. In some embodiments, fewer than 25% of the particles have a diameterin excess of 300 nm. In some embodiments, fewer than 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%or less of the particles have a diameter in excess of 300 nm.Furthermore, in some embodiments, the nanoparticles in inventivenanoparticle compositions have diameters within the range of 10 nm to300 nm.

In some embodiments, inventive nanoparticle compositions aresubstantially free of particles having a diameter in excess of 200 nm.Specifically, in some embodiments, fewer than 50%, of the nanoparticlesin inventive nanoparticle compositions have a diameter in excess of 200nm. In some embodiments, fewer than 25% of the particles have a diameterin excess of 200 nm. In some embodiments, fewer than 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%or less of the particles have a diameter in excess of 200 nm.Furthermore, in some embodiments, the nanoparticles in inventivenanoparticle compositions have diameters within the range of 10 nm to200 nm.

In some embodiments, inventive nanoparticle compositions aresubstantially free of particles having a diameter in excess of 120 nm.Specifically, in some embodiments, fewer than 50%, of the nanoparticlesin inventive nanoparticle compositions have a diameter in excess of 120nm. In some embodiments, fewer than 25% of the particles have a diameterin excess of 120 nm. In some embodiments, fewer than 20%, 19%, 18%, 17%,16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%or less of the particles have a diameter in excess of 120 nm.Furthermore, in some embodiments, the nanoparticles in inventivenanoparticle compositions have diameters within the range of 10 nm to120 nm.

In some embodiments, a majority of the nanoparticles within inventivecompositions have diameters below a specified size or within a specifiedrange. In some embodiments, the majority is more than 50%, 60%, 70%,75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%,99.9% or more of the particles in the composition.

Zeta potential is a measurement of the electric potential at a shearplane. A shear plane is an imaginary surface separating a thin layer ofliquid bound to a solid surface (e.g. the surface of inventivenanoparticles) and showing elastic behavior from the rest of liquid(e.g. liquid dispersion medium) showing normal viscous behavior. In someembodiments, inventive nanoparticles have a zeta potential rangingbetween −50 mV to +50 mV. In some embodiments, inventive nanoparticleshave a zeta potential ranging between −25 mV to +25 mV. In someembodiments, inventive nanoparticles have a zeta potential rangingbetween −10 mV to +10 mV.

In some embodiments inventive nanoparticle compositions are emulsions ordispersions. In general, an emulsion or dispersion is formed from atleast two immiscible materials, one of which will constitute thedispersion medium (i.e., the liquid medium in which particles (e.g.,nanoparticles, which constituted the “dispersed medium”) are dispersed.An “oil-in-water” dispersion is one in which oily particles aredispersed within an aqueous dispersion medium. A “water-in-oil”dispersion is one in which aqueous particles are dispersed within anoily dispersion medium. Those of ordinary skill in the art willappreciate that a dispersion can be formed from any two immiscible mediaand is not limited strictly to combinations of aqueous and oily media.The term “dispersion medium” therefore applies broadly to any dispersionmedium notwithstanding that it is common to refer to “aqueous” and“oily” categories. For example, emulsions or dispersions can be preparedfrom immiscible sets of hydrophobic/hydrophilic materials;polar/nonpolar materials, etc., regardless of whether such materials arestrictly speaking “aqueous” or “oily.”

In some embodiments, inventive nanoparticle compositions comprisemicellar structures (e.g., the nanoparticles are micelles). In someembodiments, such micellar structures are crosslinked. In someembodiments, such micellar structures are not crosslinked.

In some embodiments, inventive nanoparticle compositions self-assemblefrom a collection of combined components. In some embodiments, inventivenanoparticle compositions are prepared by subjecting a combination ofcomponents (i.e., a “premix”) to high shear force. In some embodiments,high shear force is applied by high pressure, by cavitation, byhomogenization, and/or by microfluidization. In some embodiments,combined nanoparticle-forming components are agitated, stirred, orotherwise mixed. In some such embodiments, the components are subjectedto high shear force after having been mixed. In some specificembodiments, mixing may be performed for a period of time such as, forexample, less than one hour or more than 5, 6, 7, 8, 9, 10, 11, 12, 13,14, or 15 hours. In some embodiments, solubilization is achieved.

In some embodiments of the invention, production of nanoparticlecompositions involves dialyzing a collection of components, for exampleto remove any organic solvent, and/or freeze-drying to produce acomposition.

In some embodiments of the present invention that utilize a premix, itis to be understood that the premix components may assemble intoparticles before the application of high shear force. At least some ofsuch particles may be microparticles or even nanoparticles. In someembodiments, an inventive nanoparticle composition is prepared from apremix, wherein the premix is selected from the group comprising asuspension or a microemulsion. In some embodiments, however, particlestructures do not form in the premix before application of high shearforce.

In some embodiments of the present invention, all of the componentspresent in the final nanoparticle composition are present in the premixand are subjected to high shear force to produce the nanoparticlecomposition. In some embodiments of the present invention, one or moreof the components that are present in the final nanoparticle compositionis/are missing from the premix or is/are present in the premix in asmaller amount than in the final nanoparticle composition. That is, insome embodiments of the present invention, one or more materials areadded to the nanoparticle composition after the premix is subjected tohigh shear stress.

In certain embodiments of the invention, the premix is prepared as asolution prior to application of high shear force. In particular, fornanoparticle compositions that include at least one biologically activeagent (e.g., an unmodified peptide), it is often desirable for thebiologically active agent to be dissolved in the premix before the highshear stress is applied. Thus, in many embodiments, the biologicallyactive agent is soluble in at least one of the media (or in acombination of media utilized in the premix). In some embodiments of theinvention, such dissolution requires heating; in other embodiments itdoes not.

In some embodiments of the invention, nanoparticle compositions areprepared from components including one or more aqueous, polar, orhydrophilic medium(a), one or more oily, nonpolar, or hydrophobicmedium(a), one or more micelle components, one or more surfactants oremulsifiers, one or more biologically active agents and/or one or morerelease retarding agents, etc.

Those of ordinary skill in the art will be well aware of suitableaqueous media that can be used as dispersion media or as media to bedispersed in accordance with the present invention. Representative suchaqueous media include, for example, water, saline solutions (includingphosphate buffered saline), water for injection, short chain alcohols,5% dextrose, Ringer's solutions (lactated Ringer's injection, lactatedRinger's plus 5% dextrose injection, acylated Ringer's injection),Normosol-M, Isolyte E, and the like, and combinations thereof.

Those of ordinary skill in the art will also be well aware of suitableoily media that can be used as dispersion media or as media to bedispersed in accordance with the present invention. In some embodiments,the oil may comprise one or more fatty acid groups or salts thereof. Insome embodiments, the fatty acid group may comprise digestible, longchain (e.g., C₈-C₅₀), substituted or unsubstituted hydrocarbons. In someembodiments, the fatty acid group may be a C₁₀-C₂₀ fatty acid or saltthereof. In some embodiments, the fatty acid group may be a C₁₅-C₂₀fatty acid or salt thereof. In some embodiments, the fatty acid groupmay be a C₁₅-C₂₅ fatty acid or salt thereof. In some embodiments, thefatty acid group may be unsaturated. In some embodiments, the fatty acidgroup may be monounsaturated. In some embodiments, the fatty acid groupmay be polyunsaturated. In some embodiments, a double bond of anunsaturated fatty acid group may be in the cis conformation. In someembodiments, a double bond of an unsaturated fatty acid may be in thetrans conformation.

In some embodiments, the fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, the fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linolenic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, the oil is a liquid triglyceride. In certainembodiments, the oil is a medium chain (e.g., 6-12 carbons) triglyceride(e.g., Labrafac WL 1349, coconut oil, palm kernel oil, camphor treedrupe oil, etc.). In certain embodiments, the oil is a short chain(e.g.,2-5 carbons) triglyceride. In certain embodiments, the oil is a longchain (e.g., greater than 12 carbons) triglyceride (e.g., soybean oil,sunflower oil, etc.).

Suitable oils for use with the present invention include, but are notlimited to, almond, apricot kernel, avocado, babassu, bergamot, blackcurrent seed, borage, cade, camomile, canola, caraway, carnauba, castor,cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed,emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd,grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut,lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mangoseed, meadowfoam seed, mineral, mink, nutmeg, olive, orange, orangeroughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkinseed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana,savoury, sea buckthorn, sesame, shea butter, silicone, soybean,sunflower, tea tree, thistle, tsubaki, vetiver, walnut, wheat germ, andmixtures thereof. Suitable synthetic oils for use with the presentinvention include, but are not limited to: caprylic/capric triglyceride,cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate,octyldodecanol, oleyl alcohol, and combinations thereof.

Appropriate micelle components may include, for example, one or moreamphiphilic entities. Useful amphiphilic entities include naturalentities, synthetic entities, and entities that contain both natural andsynthetic components. In some embodiments, amphiphilic entities maycomprise one or more polymers, and/or one or more compounds withpolymeric character.

In general, an amphiphilic entity is one that has both hydrophobic andhydrophilic natures. As will be appreciated by those of ordinary skillin the art, an amphiphilic entity can be comprised in any number ofdifferent ways. In some embodiments, an amphiphilic entity may compriseone or more individual compounds or molecules that is itselfamphiphilic. To give but a few examples, such compounds or moleculesinclude polyethylene glycol (PEG), phospholipids, cholesterols,glycolipids fatty acids, bile acids, and saponins PEG is generallyrecognized as safe for use in food, cosmetics, and medicines by the USFood and Drug Administration. PEG is water-soluble, non-toxic, odorless,lubricating, nonvolatile, and nonirritating.

In some embodiments, an amphiphilic entity may comprise one or moreindividual components that is not itself amphiphilic but that has somehydrophilic or hydrophobic character. In such embodiments, two or moresuch non-amphiphilic components will typically be associated with oneanother such that the assemblage of the individual components isamphiphilic. Such association may or may not involve covalent linkage;such association may involve non-covalent bonding (e.g., viaelectrostatic interactions, affinity interactions, hydrophobicinteractions, hydrogen bonding, Van der Waals interactions, ionicinteraction, dipole-dipole interaction, etc.). In general, suchassociation may involve any relevant force, bond, or means of adhesion.

In some embodiments, an amphiphilic entity for use in accordance withthe present invention may be constructed from two or more individualcomponents having differing degrees of hydrophilicity or hydrophobicity.In certain embodiments, an amphiphilic entity may comprise at least onehydrophilic component and at least one hydrophobic component. In certainembodiments, the “hydrophilic” and “hydrophobic” components are eitherhydrophilic or hydrophobic relative to one another.

In some embodiments, two or more components of differing degrees ofhydrophilicity or hydrophobicity may be bonded together by covalentbonds to form a homopolymer or a co-polymer. In some embodiments, aco-polymer may be a block co-polymer. In some embodiments, a co-polymermay be a graft co-polymer.

In some embodiments, an amphiphilic entity may comprise or consist of anamphiphilic block co-polymer. In some embodiments, an amphiphilic blockco-polymer may be a diblock co-polymer. In certain embodiments, anamphiphilic diblock co-polymer may comprise a first polymer block and asecond polymer block connected covalently at the chain ends. In specificembodiments, the first polymer block may comprise repeating units of ahydrophilic component, and the second polymer block may compriserepeating units of a hydrophobic component. In specific embodiments, thefirst polymer block may comprise repeating units of a hydrophobiccomponent, and the second polymer block may comprise repeating units ofa hydrophilic component. In some embodiments, an amphiphilic blockco-polymer may be a multiblock co-polymer. In certain embodiments, anamphiphilic block co-polymer may comprise multiple alternating blocks oftwo or more polymers connected covalently at the chain ends. In specificembodiments, an amphiphilic block co-polymer may comprise multiplealternating hydrophilic blocks and hydrophobic blocks connectedcovalently at the chain ends. In specific embodiments, each block of thealternating blocks may comprise repeating units of either hydrophiliccomponents or hydrophobic components.

In some embodiments, an amphiphilic entity may comprise or consist of anamphiphilic graft co-polymer. In some embodiments, an amphiphilic graftco-polymer may comprise or consist of blocks of polymers connectedcovalently to the side chains of other blocks of polymers. In specificembodiments, each polymer block may comprise or consist of repeatingunits of either hydrophilic or hydrophobic components. In certainembodiments, an amphiphilic graft co-polymer may comprise or consist ofa first polymer block and a second polymer block connected covalently toa side chain of the first polymer block. In certain embodiments, thefirst polymer block may comprise or consist of repeating units of ahydrophilic component, and the second block may comprise repeating unitsof a hydrophobic component. In certain embodiments, the first polymerblock may comprise or consist of repeating units of a hydrophobiccomponent, and the second block may comprise repeating units of ahydrophilic component.

In some embodiments, an amphiphilic block or graft co-polymer mayinclude a hydrophilic polymer block comprising repeating units of apolysaccharide and a hydrophobic polymer block comprising repeatingunits of a polyester or polysaccharide. Alternatively or additionally,an amphiphilic block or graft co-polymer may include a hydrophobicpolymer block comprising repeating units of a polysaccharide and ahydrophilic polymer block comprising repeating units of a polyester orpolysaccharide. Such a hydrophilic polymer block can contain repeatingunits of any type of hydrophilic polymer, such as a polysaccharide (e.g.pullulan) or polyalkene oxide (e.g. polyethylene oxide). The hydrophobicpolymer block can contain repeating units of any type of hydrophobicpolymer, such as a polycaprolactone or polyamide (e.g. polycaprolactam).

In some embodiments, the hydrophilic portion of the amphiphilic entitymay be non-ionic. In some embodiments, the hydrophilic component of anamphiphilic entity comprises one or more ionic groups. In general, suchionic groups are hydrophilic and can confer hydrophilic nature on theamphiphilic entity.

In some embodiments, the ionic group may be cationic. In someembodiments, the cationic group may be an ammonium (NH₄ ⁺), nitronium(NO₂ ⁺), nitrosyl (NO⁺), hydronium (H₃O⁺), mercurous (Hg₂ ²⁺),phosphonium (PH₄ ⁺), vanadyl (VO²⁺), or salt thereof.

In some embodiments, the ionic group may be anionic. In someembodiments, the anionic group may be a fatty acid, arsenide (As³⁻),azide (N³⁻), bromide (Br⁻), chloride (Cl⁻), fluoride (F⁻), hydride (H⁻),iodide (I⁻), nitride (N³⁻), oxide (O²⁻), phosphide (P³⁻), selenide(Se²⁻), sulfide (S²⁻), peroxide (O₂ ²⁻), arsenate (AsO₄ ³⁻), arsenite(AsO₃ ³⁻), borate (BO₃ ³⁻), perbromate (BrO₄ ⁻), bromate (BrO₃ ⁻),bromite (BrO₂ ⁻), hypobromite (BrO⁻), carbonate (CO₃ ²⁻), hydrogencarbonate (HCO₃ ⁻), chlorate (ClO₃ ⁻), perchlorate (ClO₄ ⁻), chlorite(ClO₂ ⁻), hypochlorite (ClO⁻), chromate (CrO₄ ²⁻), dichromate (Cr₂O₇²⁻), perfluorate (BrO₄ ⁻), fluorate (BrO₃ ⁻), fluorite (BrO₂ ⁻),hypofluorite (BrO⁻), periodate (lO₄ ⁻), iodate (lO₃ ⁻), iodite (lO₂ ⁻),hypoiodite (lO⁻), nitrate (NO₃ ⁻), nitrite (NO₂), phosphate (PO₄ ³),hydrogen phosphate (HPO₄ ²), dihydrogen phosphate (H2PO₄ ⁻), phosphite(PO₃ ³⁻), silicate (SiO₃ ²⁻), sulfate (SO₄ ²⁻), thiosulfate (S₂O₃ ²⁻),hydrogen sulfate (HSO₄ ⁻), sulfite (SO₃ ²⁻), hydrogen sulfite (HSO₃ ⁻),sulfonate (—S(═O)₂—O), acetate (C₂H₃O₂), formate (HCO₂), oxalate (C₂O₄²), hydrogen oxalate (HC₂O₄), citrate (C₆H_(S)O₇ ³⁻), succinate (C₄H₄O₄²⁻), fumarate (C₄H₂O₄ ²⁻), malate (C₄H_(S)O₅ ²⁻), hydrogen sulfide(HS⁻), telluride (Te²⁻), amide (NH₂ ⁻), cyanate (OCN⁻), thiocyanate(SCN⁻), cyanide (CN⁻), hydroxide (OH⁻), permanganate (MnO₄ ⁻), or saltthereof.

In some embodiments, the hydrophilic component of an amphiphilic entitymay comprise or consist of a nucleic acid. For example, the nucleic acidpolymer may include DNA, RNA, or combinations thereof. In someembodiments, the nucleic acid polymer may be an oligonucleotide and/orpolynucleotide. In some embodiments, the nucleic acid polymer may be anoligonucleotide and/or modified oligonucleotide; an antisenseoligonucleotide and/or modified antiscnsc oligonucleotide; a cDNA; agenomic DNA; viral DNA and/or RNA; DNA and/or RNA chimeras; plasmids;cosmids; gene fragments; an artificial and/or natural chromosome (e.g. ayeast artificial chromosome) and/or a part thereof; an RNA (e.g. anmRNA, a tRNA, an rRNA and/or a ribozyme); a peptide nucleic acid (PNA);a polynucleotide comprising synthetic analogues of nucleic acids, whichmay be modified or unmodified; various structural forms of DNA includingsingle-stranded DNA, double-stranded DNA, supercoiled DNA and/ortriple-helical DNA; Z-DNA; and/or combinations thereof.

In some embodiments, the hydrophilic component of an amphiphilic entitymay comprise or consist of a carbohydrate. In some embodiments, thecarbohydrate may be a polysaccharide composed of simple sugars (or theirderivatives) connected by glycosidic bonds, as known in the art. Suchsugars may include, but are not limited to, glucose, fructose,galactose, ribose, lactose, sucrose, maltose, trehalose, cellbiose,mannose, xylose, arabinose, glucoronic acid, galactoronic acid,mannuronic acid, glucosamine, galatosamine, and neuramic acid. In someembodiments, the polymer may be a hydrophilic carbohydrate, includingaminated, carboxylated, and sulfated polysaccharides. In someembodiments, the hydrophilic carbohydrate may be one or more ofpullulan, cellulose, microcrystalline cellulose, hydroxypropylmethylcellulose, hydroxycellulose, methylcellulose, dextran,cyclodextran, glycogen, starch, hydroxyethyl starch, carageenan, glycon,amylose, chitosan, N,O-carboxylmethylchitosan, algin and alginic acid,starch, chitin, heparin, konjac, glucommannan, pustulan, heparin,hyaluronic acid, curdlan, and xanthan. In some embodiments, hydrophilicpolysaccharides can be modified to become hydrophobic by introducing alarge number of side-chain hydrophobic groups. In some embodiments, ahydrophobic carbohydrate may include cellulose acetate, pullulanacetate, konjac acetate, amylose acetate, and dextran acetate.

In some embodiments, the hydrophilic component of an amphiphilic entitymay comprise or consist of a gum including, but not limited to, xanthangum, alginic acid, caraya gum, sodium alginate, and/or locust bean gum.

In some embodiments, a component of an amphiphilic entity may compriseor consist of a protein. In some embodiments, a protein is a hydrophiliccomponent of an amphiphilic entity. In other embodiments, a protein is ahydrophobic component of an amphiphilic entity. Exemplary proteins thatmay be used in accordance with the present invention include, but arenot limited to, albumin, collagen, or a poly(amino acid) (e.g.polylysine).

In some embodiments, the hydrophobic component of an amphiphilic entitymay comprise or consist of one or more fatty acid groups or saltsthereof. In general, such groups are typically hydrophobic and canconfer hydrophobic nature onto the amphiphilic entity. In someembodiments, the fatty acid group may comprise digestible, long chain(e.g., C₈-C₅₀), substituted or unsubstituted hydrocarbons. In someembodiments, the fatty acid group may be a C₁₀-C₂₀ fatty acid or saltthereof. In some embodiments, the fatty acid group may be a C₁₅-C₂₀fatty acid or salt thereof. In some embodiments, the fatty acid groupmay be a C₁₅-C₂₅ fatty acid or salt thereof. In some embodiments, thefatty acid group may be unsaturated. In some embodiments, the fatty acidgroup may be monounsaturated. In some embodiments, the fatty acid groupmay be polyunsaturated. In some embodiments, a double bond of anunsaturated fatty acid group may be in the cis conformation. In someembodiments, a double bond of an unsaturated fatty acid may be in thetrans conformation.

In some embodiments, the fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, the fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, the hydrophobic component of an amphiphilic entitymay comprise or consist of one or more biocompatible and/orbiodegradable synthetic polymers, including, for example, polycarbonates(e.g. poly(1,3-dioxan-2one)), polyanhydrides (e.g. poly(sebacicanhydride)), polyhydroxyacids (e.g. poly(β-hydroxyalkanoate)),polypropylfumarates, polycaprolactones, polyamides (e.g.polycaprolactam), polyacetals, polyethers, polyesters (e.g. polylactideand polyglycolide), biodegradable polycyanoacrylates, polyvinylalcohols, and biodegradable polyurethanes. For example, the amphiphilicentity may comprise one or more of the following biodegradable polymers:poly(lactic acid), poly(glycolic acid), poly(caprolactone),poly(lactidc-co-glycolide), poly(lactidc-co-caprolactone),poly(glycolide-co-caprolactone), and poly(DL-lactide-co-glycolide).

In some embodiments, the hydrophobic component of an amphiphilic entitymay comprise or consist of one or more acrylic polymers. In certainembodiments, acrylic polymers include, for example, acrylic acid andmethacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethylmethacrylates, cyanoethyl methacrylate, aminoalkyl methacrylatecopolymer, poly(acrylic acid), poly(methacrylic acid), methacrylic acidalkylamide copolymer, poly(methyl methacrylate), poly(methacrylic acidanhydride), methyl methacrylate, polymethacrylate, poly(methylmethacrylate) copolymer, polyacrylamide, aminoalkyl methacrylatecopolymer, glycidyl methacrylate copolymers, and combinations comprisingone or more of the foregoing polymers. The acrylic polymer may comprisefully-polymerized copolymers of acrylic and methacrylic acid esters witha low content of quaternary ammonium groups.

In some embodiments, the hydrophobic component of an amphiphilic entitymay comprise or consist of a polyester. Exemplary such polyestersinclude, for example, polyalkylene glycols, poly(glycolide-co-lactide),PEGylated poly(lactic-co-glycolic acid), poly(lactic acid), PEGylatedpoly(lactic acid), poly(glycolic acid), PEGylated poly(glycolic acid),co-polymers of polylactic and polyglycolic acid, and derivativesthereof. In some embodiments, polyesters include, for example,polyanhydrides, poly(ortho ester) PEGylated poly(ortho ester),poly(caprolactone), PEGylated poly(caprolactone), polylysine, PEGylatedpolylysine, poly(ethylene imine), PEGylated poly(ethylene imine), andderivatives thereof. In some embodiments, polyesters may include, forexample, polycaprolactone, poly(L-lactide-co-L-lysine), poly(serineester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

Suitable surfactants or emulsifying agents include, but are not limitedto, phosphoglycerides; phosphatidylcholines; dipalmitoylphosphatidylcholine (DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoylphosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid amides; sorbitan trioleate (Span 85) glycocholate; sorbitanmonolaurate (Span 20); polysorbate 20 (Tween-20); polysorbate 60(Tween-60); polysorbate 65 (Tween-65); polysorbate 80 (Tween-80);polysorbate 85 (Tween-85); polyoxyethylene monostearate; surfactin; apoloxomer; a sorbitan fatty acid ester such as sorbitan trioleate;lecithin; lysolecithin; phosphatidylserine; phosphatidylinositol;sphingomyelin; phosphatidylethanolamine (cephalin); cardiolipin;phosphatidic acid; cerebrosides; dicetylphosphate;dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine;hexadecyl-amine; acetyl palmitate; glycerol ricinoleate; hexadecylsterate; tyloxapol; poly(ethylene glycol)5000-phosphatidylethanolamine;poly(ethylene glycol)400-monostearate; and phospholipids. The surfactantcomponent may be a mixture of different surfactants. These surfactantsmay be extracted and purified from a natural source or may be preparedsynthetically in a laboratory. In a preferred embodiment, thesurfactants are commercially available.

In certain embodiments of the invention, relative amounts of componentsutilized to prepare inventive nanoparticle compositions are selected oradjusted to generate nanoparticles having desired characteristics. Insome embodiments, the oil and surfactant are utilized at a ratio rangingbetween 0.25-10. In some embodiments, the ratio of oil to surfactant isapproximately 0.25:1, approximately 0.5:1, approximately 1:1,approximately 2:1, approximately 3:1, approximately 4:1, approximately5:1, approximately 6:1, approximately 7:1, approximately 8:1,approximately 9:1, or approximately 10:1. In some embodiments, the ratioof surfactant to oil is approximately 0.5:1, approximately 1:1,approximately 2:1, approximately 3:1, approximately 4:1, approximately5:1, approximately 6:1, approximately 7:1, approximately 8:1,approximately 9:1, or approximately 10:1. In some embodiments, the oiland surfactant are utilized at a ratio ranging between 0.25-2. In someembodiments, the ratio of oil to surfactant is approximately 0.25:1,approximately 0.5:1, approximately 1:1, or approximately 2:1. In someembodiments, the ratio of surfactant to oil is approximately 0.5:1,approximately 1:1, or approximately 2:1. In certain embodiments, theratio of oil to surfactant is approximately 1:1.

In some embodiments, the percent of oil in the composition from whichnanoparticles arc prepared (e.g., in the premix) ranges between 0% to30%. In some embodiments the percent of oil in the composition fromwhich nanoparticles are prepared (e.g., in the premix) is approximately1%, approximately 2%, approximately 3%, approximately 4%, approximately5%, approximately 6%, approximately 7%, approximately 9%, approximately10%, approximately 11%, approximately 12%, approximately 13%,approximately 14%, approximately 15%, approximately 16%, approximately17%, approximately 18%, approximately 19%, approximately 20%,approximately 21%, approximately 22%, approximately 23%, approximately24%, approximately 25%, approximately 26%, approximately 27%,approximately 28%, approximately 29%, or approximately 30%. In someembodiments the percent of oil is approximately 8%. In some embodimentsthe percent of oil is approximately 5%.

In some embodiments, where one or more amphiphilic entities is/areutilized, the percent of amphiphilic entity in the composition fromwhich nanoparticles are prepared (e.g., in the premix) can range from40% to 99%, from 50% to 99%, from 60% to 99%, from 70% to 99%, from 80%to 99%, from 80% to 90%, or from 90% to 99%. In some embodiments thepercent of amphiphilic entity in the composition from whichnanoparticles are prepared (e.g., in the premix) is approximately 75%,approximately 76%, approximately 77%, approximately 78%, approximately79%, approximately 80%, approximately 81%, approximately 82%,approximately 83%, approximately 84%, approximately 85%, approximately86%, approximately 87%, approximately 88%, approximately 89%,approximately 90%, approximately 91%, approximately 92%, approximately93%, approximately 94%, approximately 95%, approximately 96%,approximately 97%, approximately 98%, or approximately 99%.

The percent of substances with surfactant activity in the premix canrange from 0% to 99%, from 10% to 99%, from 25% to 99%, from 50% to 99%,or from 75% to 99%. In some embodiments, the percent of substances withsurfactant activity in the premix can range from 0% to 75%, from 0% to50%, from 0% to 25%, or from 0% to 10%. In some embodiments, the percentof surfactant in the composition from which nanoparticles are prepared(e.g., in the premix) ranges between 0% -30%. In some embodiments thepercent of surfactant in is approximately 1%, approximately 2%,approximately 3%, approximately 4%, approximately 5%, approximately 6%,approximately 7%, approximately 9%, approximately 10%, approximately11%, approximately 12%, approximately 13%, approximately 14%,approximately 15%, approximately 16%, approximately 17%, approximately18%, approximately 19%, approximately 20%, approximately 21%,approximately 22%, approximately 23%, approximately 24%, approximately25%, approximately 26%, approximately 27%, approximately 28%,approximately 29%, or approximately 30%. In some embodiments the percentof surfactant is approximately 8%. In some embodiments the percent ofsurfactant is approximately 5%.

In some embodiments, the nanoparticle composition does not contain morethan one oil. In some embodiments, the nanoparticle composition maycomprise two or more oils. In some embodiments, the nanoparticlecomposition does not contain more than one surfactant. In someembodiments, the nanoparticle composition may comprise two or moresurfactants. In some embodiments, the nanoparticle composition iscompletely free or substantially free of toxic components.

In some embodiments, the nanoparticle composition consists essentiallyof water, an oil, a surfactant, and at least one biologically activeagent (e.g., and unmodified peptide). In some embodiments, thenanoparticle composition consists essentially of water, an oil, asurfactant, at least one biologically active agent, and at least onesubstance used to produce and/or preserve the nanoparticle composition.

In some embodiments, the nanoparticle composition consists of water, anoil, a surfactant, and an unmodified peptide. In some embodiments, thenanoparticle composition consists of water, an oil, a surfactant, anunmodified peptide, and at least one substance used to produce and/orpreserve the nanoparticle.

Unmodified Peptides

Any of a variety of peptides may be incorporated in nanoparticlecompositions according to the present invention. In most embodiments, ita peptide is less than about 100 amino acids in length; in someembodiments, a peptide is less than about 90, about 80, about 70, about65, about 60, about 55, about 50, about 45, about 40, about 35, about30, about 25, about 20, about 15, about 13, about 12, about 10, about 9,about 8, about 7, about 6, or about 5 amino acids in length. In somespecific embodiments, the peptide is a penta peptide. In someembodiments, a peptide to be incorporated in a nanoparticle compositionsis comprised solely of naturally occurring amino acids. In someembodiments, a peptide comprises one or more non-naturally occurringamino acid.

Unmodified short peptides for use in accordance with the presentinvention, generally, are ones that have biological activity in the skin(including epidermis and dermis), sub-cutaneous tissue (includingadipose tissue) and/or contiguous muscles. Such peptides include, butare not limited to, peptides to promote extra-cellular matrix production(e.g., KTTKS, SEQ ID NO.: 1; EYKTTKSSRL, SEQ ID NO.: 2; VIEYKTTK, SEQ IDNO.: 3; KTTK, SEQ ID NO.: 4; GKTVIEYKTTKS, SEQ ID NO.: 5;GKTVIEYKTTKSSRL, SEQ ID NO.: 6; WGKTVIEYKTTKSSRLPIID, SEQ ID NO.: 7;CTSHTGAWGKTVIEYKTTKS, SEQ ID NO.: 8; TTKS, SEQ ID NO.: 9), peptides thatmay decrease wrinkles (e.g., EEMQRR, SEQ ID NO.: 10), peptides toimprove wound healing (e.g., gastrin-releasing peptide, VGVAPG, SEQ IDNO.: 11; YYRADA, SEQ ID NO.: 12; GHK, SEQ ID NO.: 13, interferon,interferon inducer), and peptides (e.g., P144; TSLDASIIWAMMQN, SEQ IDNO.: 14) to treat excessive accumulation of extra-cellular matrix thatare result in conditions such as hypertrophic scarring, keloids, andlocalized or systemic sclerosis (scleroderma) (Katayama, et al.; supra,Lupo, supra; Robinson et al., supra; Bhartiya et al., 1992, J. Cell.Physiol., 150:312; and Santiago et al., 2005, J. InvestigativeDermatology, 125:450; all of which are incorporated herein byreference). See Table 1 below for definitions of peptide abbreviations.

TABLE I Peptide Abbreviations Trivial Systematic name^(a) Symbols^(b)Name^(c) Formula Alanine Ala A 2-Aminopropanoic acid CH₃—CH(NH₂)—COOHArginine Arg R 2-Amino-5- H₂N—C(═NH)—NH—[CH₂]₃—CH(NH₂)—COOHguanidinopentanoic acid Asparagine Asn^(d) N^(d) 2-Amino-3-H₂N—CO—CH₂—CH(NH₂)—COOH carbamoylpropanoic acid Aspartic acid Asp^(d)D^(d) 2-Aminobutanedioic HOOC—CH₂—CH(NH₂)—COOH acid Cysteine Cys C2-Amino-3- HS—CH₂—CH(NH₂)—COOH mercaptopropanoic acid Glutamine Gln^(d)Q^(d) 2-Amino-4- H₂N—CO—[CH₂]₂—CH(NH₂)—COOH carbamoylbutanoic acidGlutamic Glu^(d) E^(d) 2-Aminopentanedioic acid HOOC—[CH₂]₂—CH(NH₂)—COOHacid Glycine Gly G Aminoethanoic acid CH₂(NH₂)—COOH Histidine His H2-Amino-3-(1H-imidazol-4- yl)-propanoic acid

Isolcucinc Ilc I 2-Amino-3-methylpentanoic C₂H₅—CH(CH₂)—CH(NH₂)—COOHacid^(e) Lcucinc Lcu L 2-Amino-4-methyl- (CH₃)₂CH—CH₂—CH(NH₂)—COOHpentanoic acid Lysine Lys K 2,6-Diaminohexanoic acidH₂N—[CH₂]₄—CH(NH₂)—COOH Methionine Met M 2-Amino-4-CH₃—S[CH₂]₂—CH(NH₂)—COOH (methylthio)butanoic acid Phenylalanine Phe F2-Amino-3-phenyl- C₆H₅—CH₂—CH(NH₂)—COOH propanoic acid Proline Pro PPyrrolidine-2-carboxylic acid

Serine Ser S 2-Amino-3- HO—CH₂—CH(NH₂)—COOH hydroxypropanoic acidThreonine Thr T 2-Amino-3-hydroxy- CH₃—CH(OH)—CH(NH₂)—COOH butanoicacid^(c) Tryptophan Trp W 2-Amino-3-(1H-indol-3-yl)- propanoic acid

Tyrosine Tyr Y 2-Amino-3-(4- hydroxyphenyl)- propanoic acid

Valine Val V 2-Amino-3-methylbutanoic (CH₃)₂CH—CH(NH₂)—COOH acid

Other Components

As indicated herein, inventive nanoparticle compositions may contain orbe combined with one or more other components. Certain exemplary suchother components are discussed here.

Biologically-Active Agents

Any biologically active agents, including, for example, therapeutic,diagnostic, prophylactic, nutritional, cosmetic, and/or dermatologicalagents, may be delivered according to the present invention. Suchbiologically active agents may be small molecules, organometalliccompounds, nucleic acids, proteins (including multimeric proteins,protein complexes, etc.), peptides, lipids, carbohydrates, herbs,hormones, metals, radioactive elements and compounds, drugs, vaccines,immunological agents, etc., and/or combinations thereof. Suchbiologically agents may be encapsulated within, adsorbed to the surfaceof, present at the interface of and/or present within a micellarmembrane of inventive nanoparticles.

In some embodiments, the percent of biologically active agent in thecomposition used to prepare inventive nanoparticles (e.g., in thepremix) and/or in the nanoparticles ranges from 0.1%-25%. In someembodiments, the percentage of biologically active agent ranges from0.1%-20%, from 0.1%-15%, from 0.1%-10%, from 0.1% -5%, or from 0.1%-1%.In some embodiments, the percentage of biologically active agent rangesfrom 1%-20%, from 5%-20%, from 10%-20%, from 15%-20%, or from 15%-25%.In some embodiments, the percentage of biologically active agent is lessthan 0.1%. In some embodiments, the percentage of biologically activeagent is greater than 25%. In some embodiments, the percentage ofbiologically active agent is approximately 0.1%, approximately 0.5%,approximately 1%, approximately 2%, approximately 3%, approximately 4%,approximately 5%, approximately 6%, approximately 7%, approximately 8%,approximately 9%, approximately 10%, approximately 11%, approximately12%, approximately 13%, approximately 14%, approximately 15%,approximately 16%, approximately 17%, approximately 18%, approximately19%, approximately 20%, approximately 21%, approximately 22%,approximately 23%, approximately 24%, approximately 25%, or greater.

Relevant biologically active agents can be produced or obtainedaccording to any available method or approach. Biologically activeagents may contain, or be modified to contain, one or more moietiesintended to facilitate their use or delivery in conjunction withinventive nanoparticles. Such modification should not interfere with thebiological activity of the agent. In some embodiments, the modificationcan optionally be removed in vivo. For example, biologically activeagents may be detectably labeled and/or may be provided in a “pro” formthat is converted or modified after delivery into an active form.

In some embodiments, the biologically active agent is a small moleculeand/or organic compound with pharmaceutical activity. In someembodiments, the biologically active agent is a clinically-used drug. Insome embodiments, the drug is an antibiotic, anti-viral agent,anesthetic, anticoagulant, anti-cancer agent, inhibitor of an enzyme,steroidal agent, anti-inflammatory agent, anti-neoplastic agent,antigen, vaccine, antibody, decongestant, antihypertensive, sedative,birth control agent, progestational agent, anti-cholinergic, analgesic,anti-depressant, anti-psychotic, β-adrenergic blocking agent, diuretic,cardiovascular active agent, vasoactive agent, non-steroidalanti-inflammatory agent, etc. Of particular interest are biologicallyactive agents suitable for transdermal administration.

The biologically active agents delivered may be a mixture ofpharmaceutically active agents. For example, a local anesthetic may bedelivered in combination with an anti-inflammatory agent such as asteroid. Local anesthetics may also be administered with vasoactiveagents such as epinephrine. To give but another example, an antibioticmay be combined with an inhibitor of the enzyme commonly produced bybacteria to inactivate the antibiotic (e.g., penicillin and clavulanicacid).

In some embodiments, the biologically active agent is a diagnosticagent. In some embodiments, diagnostic agents include gases;commercially available imaging agents used in positron emissionstomography (PET), computer assisted tomography (CAT), single photonemission computerized tomography, x-ray, fluoroscopy, and magneticresonance imaging (MRI); and contrast agents. Examples of suitablematerials for use as contrast agents in MRI include gadolinium chelates,as well as iron, magnesium, manganese, copper, and chromium. Examples ofmaterials useful for CAT and x-ray imaging include iodine-basedmaterials.

In some embodiments, the biologically active agent is a prophylacticagent. In some embodiments, prophylactic agents include vaccines.Vaccines may comprise isolated proteins or peptides, inactivatedorganisms and viruses, dead organisms and virus, genetically alteredorganisms or viruses, and cell extracts. Prophylactic agents may becombined with interleukins, interferon, cytokines, and adjuvants such ascholera toxin, alum, Freund's adjuvant, etc. Prophylactic agents mayinclude antigens of such bacterial organisms as Streptococccuspnuemoniae, Haemophilus influenzae, Staphylococcus aureus, Streptococcuspyrogenes, Corynebacterium diphtheriae, Listeria monocytogenes, Bacillusanthracis, Clostridium tetani, Clostridium botulinum, Clostridiumperfringens, Neisseria meningitidis, Neisseria gonorrhoeae,Streptococcus mutans, Pseudomonas aeruginosa, Salmonella typhi,Haemophilus parainfluenzae, Bordetella pertussis, Francisellatularensis, Yersinia pestis, Vibrio cholerae, Legionella pneumophila,Mycobacterium tuberculosis, Mycobacterium leprae, Treponema pallidum,Leptospirosis interrogans, Borrelia burgdorferi, Camphylobacter jejuni,and the like; antigens of such viruses as smallpox, influenza A and B,respiratory syncytial virus, parainfluenza, measles, HIV,varicella-zoster, herpes simplex 1 and 2, cytomegalovirus, Epstein-Barrvirus, rotavirus, rhinovirus, adenovirus, papillomavirus, poliovirus,mumps, rabies, rubella, coxsackieviruses, equine encephalitis, Japaneseencephalitis, yellow fever, Rift Valley fever, hepatitis A, B, C, D, andE virus, and the like; antigens of fungal, protozoan, and parasiticorganisms such as Cryptococcus neoformans, Histoplasma capsulatum,Candida albicans, Candida tropicalis, Nocardia asteroides, Rickettsiaricketsii, Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci,Chlamydial trachomatis, Plasmodium falciparum, Trypanosoma brucei,Entamoeba histolytica, Toxoplasma gondii, Trichomonas vaginalis,Schistosoma mansoni, and the like. These antigens may be in the form ofwhole killed organisms, peptides, proteins, glycoproteins,carbohydrates, or combinations thereof.

In some embodiments, the biologically active agent may be a protein. Asused herein, the terms “protein” and “peptide” can be usedinterchangeably. In certain embodiments, peptides range from about 5 toabout 40, about 10 to about 35, about 15 to about 30, or about 20 toabout 25 amino acids in size. Peptides from panels of peptidescomprising random sequences and/or sequences which have been variedconsistently to provide a maximally diverse panel of peptides may beused.

In some embodiments, the biologically active agent may be an antibody.In some embodiments, antibodies may include, but are not limited to,polyclonal, monoclonal, chimeric (i.e. “humanized”), single chain(recombinant) antibodies. In some embodiments, antibodies may havereduced effector functions and/or bispecific molecules. In someembodiments, antibodies may include Fab fragments and/or fragmentsproduced by a Fab expression library.

In some embodiments, the biologically active agent may be a nucleicacid. In some embodiments, the oligonucleotides comprise DNA, RNA,chimeric mixtures, derivatives, characteristic portions, and/or modifiedversions thereof. The oligonucleotides of the present invention may besingle-stranded and/or double-stranded. The oligonucleotide may bemodified at the base moiety, sugar moiety, and/or phosphate backbone,for example, to improve stability of the molecule, hybridization, etc.

In specific embodiments, a nucleic acid comprises an antisense moleculethat binds to a translational start site, transcriptional start site,and/or splice junctions. Antisense oligonucleotides will bind to atarget mRNA and/or prevent translation. Alternatively or additionally,the antisense oligonucleotide may bind to DNA of a target gene, such as,for example, a regulatory element.

In some embodiments, a nucleic acid comprises a ribozyme designed tocatalytically cleave target mRNA transcripts may be used to preventtranslation of a target mRNA and/or expression of a target (see, e.g.,PCT publication WO 90/11364; and Sarver et al., 1990, Science 247:1222;both of which are incorporated herein by reference).

Alternatively or additionally, endogenous target gene expression may bereduced by targeting deoxyribonucleotide sequences complementary to theregulatory region of the target gene (i.e., the target gene's promoterand/or enhancers) to form triple helical structures that preventtranscription of the target gene in target muscle cells in the body (seegenerally, Helene, 1991, Anticancer Drug Des. 6:569; Helene et al.,1992, Ann, N.Y. Acad. Sci. 660:27; and Maher, 1992, Bioassays 14:807;all of which are incorporated herein by reference).

In some embodiments, the biologically active agent is a nutraceuticalagent. In some embodiments, the nutraceutical agent provides basicnutritional value. In some embodiments, the nutraceutical agent provideshealth or medical benefits. In some embodiments, the nutraceutical agentis a dietary supplement.

In some embodiments, the nutraceutical agent is a vitamin. In someembodiments, the vitamin is one or more of vitamin A (retinoids),vitamin B1 (thiamine), vitamin B2 (riboflavin), vitamin B3 (niacin),vitamin B5 (pantothenic acid), vitamin B6 (pyroxidone), vitamin B7(biotin),vitamin B9 (folic acid), vitamin B12 (cyanocobalamin), vitaminC (ascorbic acid), vitamin D, vitamin E, or vitamin K.

In some embodiments, the nutraceutical agent is a mineral. In someembodiments, the mineral is one or more of bismuth, boron, calcium,chlorine, chromium, cobalt, copper, fluorine, iodine, iron, magnesium,manganese, molybdenum, nickel, phosphorus, potassium, rubidium,selenium, silicon, sodium, strontium, sulfur, tellurium, titanium,tungsten, vanadium, or zinc.

In some embodiments, the nutraceutical agent is an essential amino acid.In some embodiments, the amino acid is one or more of arginine,glutamine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, threonine, tryptophan, or valine.

In some embodiments, nutraceutical agents may include fatty acids and/oromega-3 fatty acids (e.g. DHA or ARA), fruit and vegetable extracts,lutein, phosphatidylserine, lipoid acid, melatonin, glucosamine,chondroitin, aloe vera, guggul, green tea, lycopene, whole foods, foodadditives, herbs, phytonutrients, antioxidants, flavonoid constituentsof fruits, evening primrose oil, flaxseeds, fish and marine animal oils(e.g. cod liver oil), and probiotics. In some embodiments, nutraceuticalagents may include bio-engineered foods genetically-engineered to have adesired property (also known as “pharmafoods”).

Exemplary nutraceutical agents and dietary supplements are disclosed,for example, in Roberts et al., (Nutriceuticals: The CompleteEncyclopedia of Supplements, Herbs, Vitamins, and Healing Foods,American Nutriceutical Association, 2001; incorporated herein byreference). Nutraceutical agents and dietary supplements are alsodisclosed in Physicians' Desk Reference for Nutritional Supplements, 1stEd., 2001, and Physicians' Desk Reference for Herbal Medicines, 1st Ed.,2001 (both of which are incorporated herein by reference).

In some embodiments, inventive nanoparticles loaded with nutraceuticalagents can be incorporated into food substances. For example, thenutraceutical-loaded nanoparticles can be dissolved into liquids, suchas beverages.

In some embodiments, the biologically active agent is a cosmetic and/ordermatological agent. In some embodiments, the cosmetic and/ordermatological agent may include vitamins and their derivatives (e.g.vitamin E and its esters, vitamin C and its esters, vitamins B, vitaminA alcohol or retinol and its esters), provitamins (e.g. panthenol,niacinamide or ergocalciferol), antioxidants, phenolic compounds (e.g.benzoyl peroxide), essential oils, humectants, sunscreen agents,moisturizing agents, proteins, ceramides, and pseudoceramides.

In some embodiments, the biologically active agent may be one or morebotulinum toxin peptides or protein complexes. In some embodiments, thebotulinum toxin may be one or more of botulinum toxin serotypes A, B,C₁, C₂, D, E, F, or G. In some embodiments, the botulinum toxin may bean isolated and/or purified botulinum toxin. In some embodiments, thebotulinum toxin may be a partially-isolated and/or partially-purifiedbotulinum toxin. In some embodiments, the botulinum toxin may be anative botulinum complex. In some embodiments, the botulinum toxin maybe associated with non-toxin proteins. In some embodiments, thebotulinum toxin may be a recombinantly-made botulinum toxin.

Those skilled in the art will recognize that this is an exemplary, notcomprehensive, list of biologically active agents. Any biologicallyactive agent may be encapsulated within or bound to the surface ofnanoparticles.

Release Retarding Agents

In some embodiments of the invention, particularly those containing oneor more biologically active agents (e.g., unmodified peptides),inventive nanoparticle compositions further include or are formulatedwith one or more release-retarding ingredients to allow for controlledrelease of the agent. Any release-retarding ingredient known in the artis suitable for use in making the inventive nanoparticles. In someembodiments, release-retarding ingredients are hydrophilic and/orhydrophobic polymers. Release-retarding ingredients include, for examplecelluloses or derivatives thereof, acrylic polymers, ester polymers,vinyl-pyrrolidone-based polymers, gums, other natural polymers, and/orcombinations of these.

In some embodiments, the release-retarding ingredient is cellulose or aderivative thereof. In certain embodiments, the cellulose or derivativethereof comprises one or more of hydroxypropyl methyl cellulose,methylcellulose, carboxymethyl cellulose, sodium carboxymethylcellulose,hydroxypropyl ethylcellulose, hydroxyethylcellulose, and hydroxypropylcellulose. In certain embodiments, the cellulose or derivative thereofis methylcellulose or a derivative thereof. In certain embodiments, thecellulose or derivative thereof is hydroxypropyl methylcellulose (HPMC).Those skilled in the art will appreciate that other cellulosic polymers,including other alkyl cellulosic polymers, can be utilized.

In some embodiments, the release-retarding ingredient is an acrylicpolymer. In certain embodiments, acrylic polymers include, for example,acrylic acid and methacrylic acid copolymers, methyl methacrylatecopolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate,aminoalkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylicacid), methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers, andcombinations comprising one or more of the foregoing polymers. Theacrylic polymer may comprise fully polymerized copolymers of acrylic andmethacrylic acid esters with a low content of quaternary ammoniumgroups.

In some embodiments, the release-retarding ingredient is a polyester. Insome embodiments, polyesters include polyalkylene glycols,poly(glycolide-co-lactide), PEGylated poly(lactic-co-glycolic acid),poly(lactic acid), PEGylated poly(lactic acid), poly(glycolic acid),PEGylated poly(glycolic acid), co-polymers of polylactic andpolyglycolic acid, and derivatives thereof. In some embodiments,polyesters include, for example, polyanhydrides, poly(ortho ester)PEGylated poly(ortho ester), poly(caprolactone), PEGylatedpoly(caprolactone), polylysine, PEGylated polylysine, poly(ethyleneimine), PEGylated poly(ethylene imine), and derivatives thereof. In someembodiments, polyesters include, for example, polycaprolactone,poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof.

In some embodiments, the release-retarding ingredient is a cross-linkedpolymer of poly(vinyl-pyrrolidone). In some embodiments, the polymer iscrosspovidone. In some embodiments, the polymer is un-cross-linkedpoly(vinyl-pyrrolidone). In some embodiments, the polymer is povidone.

In some embodiments, the release-retarding ingredient may be a naturalpolymer. In some embodiments, the natural polymer is a gum, including,for example, xanthan gum, alginic acid, caraya gum, sodium alginate,and/or locust bean gum. In some embodiments, the natural polymer may bea protein (e.g. albumin), lipid, nucleic acid, or carbohydrateiments,the release-retarding ingredient is a polyester. In some embodiments,polyesters include polyalkylene glycols, poly(glycolide-co-lactide),PEGylated poly(lactic-co-glycolic acid), poly(lactic acid), PEGylatedpoly(lactic acid), poly(glycolic acid), PEGylated poly(glycolic acid),co-polymers of polylactic and polyglycolic acid, and derivativesthereof. In some embodiments, polyesters include, for example,polyanhydrides, poly(ortho ester) PEGylated poly(ortho ester),poly(caprolactone), PEGylated poly(caprolactone), polylysine, PEGylatedpolylysine, poly(ethylene imine), PEGylated poly(ethylene imine), andderivatives thereof. In some embodiments, polyesters include, forexample, polycaprolactone, poly(L-lactide-co-L-lysine), poly(serineester), poly(4-hydroxy-L-proline ester),poly[α-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In some embodiments, the release-retarding ingredient is a cross-linkedpolymer of poly(vinyl-pyrrolidone). In some embodiments, the polymer iscrosspovidone. In some embodiments, the polymer is un-cross-linkedpoly(vinyl-pyrrolidone). In some embodiments, the polymer is povidone.In some embodiments, the release-retarding ingredient may be a naturalpolymer. In some embodiments, the natural polymer is a gum, including,for example, xanthan gum, alginic acid, caraya gum, sodium alginate,and/or locust bean gum. In some embodiments, the natural polymer may bea protein (e.g. albumin), lipid, nucleic acid, or carbohydrate

Formulating Agents

Inventive nanoparticle compositions may be formulated for administrationto a subject. In certain embodiments, inventive nanoparticlecompositions are formulated for application to the skin, to achievetransdermal delivery to the subject. For example, inventive nanoparticlecompositions may be formulated in cosmetic or other preparationsintended to be topically applies.

Human skin comprises the dermis and the epidermis. The epidermis hasseveral layers of tissue, namely, stratum comeum, stratum lucidum,stratum granulosum, stratum spinosum, and stratum basale (identified inorder from the outer surface of the skin inward). The stratum comeumpresents the most significant hurdle in transdermal delivery ofmedications generally, and presumably of unmodified peptides inparticular. The stratum comeum is typically about 10-15 μm thick, and itconsists of flattened, keratised cells (corneocytes) arranged in severallayers. The intercellular space between the corneocytes is filled withlipidic structures, and may play an important role in the permeation ofsubstances through skin (Bauerova et al., 2001, European Journal of DrugMetabolism and Pharmacokinetics, 26:85; incorporated herein byreference).

The rest of the epidermis below the stratum corneum is approximately 150μm thick. The dermis is about 1 mm-2 mm thick and is located below theepidermis. The dermis is innervated by various capillaries as well asneuronal processes.

Traditionally, attempts at transdermal administration of medication havebeen focused in increasing the permeability of the stratum comeum. Someattempts have included using chemical enhancing agents that increase thepermeability of molecules through the skin. Some attempts have includedusing mechanical apparatus to bypass or ablate portions of the stratumcorneum. In addition, attempts have included use of ultrasound oriontophoresis to facilitate the permeation of pharmaceuticals throughthe skin. In most cases, the goal has been to a pharmaceutical agent,typically a small molecule, through the skin, typically so that an agentmay pass to the capillary bed in the dermis where the agent may besystemically incorporated into the subject to achieve a therapeuticeffect.

The present invention provides, among other things, methods ofadministering unmodified peptides transdermally that do not require useof abrasive or other disrupting agents (whether chemical, mechanical,electrical, magnetic, etc.). Rather, the present inventors havesurprisingly found that botulinum toxin incorporated into inventivenanoparticle compositions is effectively delivered transdermally withoutfurther steps to permeabilize or disrupt the stratum corneum. Use ofsuch agents or steps with inventive botulinum nanoparticle compositionsis not necessarily precluded in all embodiments of the presentinvention, but also is not required.

The present invention therefore provides methods of administeringunmodified peptides through the topical application of an inventivenanoparticle composition. In some embodiments, the inventivenanoparticle composition is applied directly to the skin and forabsorption through the epidermal layers. In some embodiments, thenanoparticle composition can penetrate the top layer of the skin,including the stratum corneum, dermal pores, and/or dermal glands,without the use of chemical or mechanical skin permeation enhancers orother agents that cause abrasion.

It will be appreciated by those of ordinary skill in the art thatinventive compositions for topical administration may be prepared as acosmetic formulation such as skin softener, nutrition lotion typeemulsion, cleansing lotion, cleansing cream, skin milk, emollientlotion, massage cream, emollient cream, make-up base, lipstick, facialpack or facial gel, cleaner formulation such as shampoos, rinses, bodycleanser, hair-tonics, or soaps, or dermatological composition such aslotions, ointments, gels, creams, patches or sprays.

Such formulation of inventive nanoparticle compositions typicallyincludes combination with one or more excipients such as, for example,fillers, sequestering agents, softeners, coloring materials (e.g.pigments and dyes), and fragrances.

In some embodiments, inventive nanoparticle compositions are formulatedas a cream. The term “cream” refers to a spreadable composition,typically formulated for application to the skin Creams typicallycontain an oil and/or fatty acid based-matrix. Creams formulatedaccording to the present invention may contain nanoparticles and may becapable of substantially complete penetration (e.g., of suchnanoparticles) through the skin upon topical administration. Such acream could also act as a carrier for incorporated materials (e.g., forexample, for a botulinum toxin).

Those of ordinary skill in the art will appreciate that inventivenanoparticle compositions may be incorporated into a device such as, forexample, a patch.

A variety of transdermal patch structures are known in the art; those ofordinary skill will appreciate that inventive nanoparticle compositionsmay readily be incorporated into any of a variety of such structures. Insome embodiments, a transdermal patch may further comprise a pluralityof needles extending from one side of the patch that is applied to theskin, wherein the needles extend from the patch to project through thestratum comeum of the skin. In some embodiments, the needles do notrupture a blood vessel.

In some embodiments of the present invention, a nanoparticle compositioncan be provided in a depot in a patch so that pressure applied to thepatch causes unmodified peptide to be directed out of the patch(optionally through needles) and through the stratum comeum.

In some embodiments of the present invention, a transdermal patchincludes an adhesive. Some examples of adhesive patches are well known(for example, see U.S. Pat. Des. Nos. 296,006; 6,010,715; 5,591,767;5,008,110; 5,683,712; 5,948,433; and 5,965,154; all of which areincorporated herein by reference). Adhesive patches are generallycharacterized as having an adhesive layer, which will be applied to aperson's skin, a depot or reservoir for holding a pharmaceutical agent,and an exterior surface that prevents leakage of the pharmaceutical fromthe depot. The exterior surface of a patch is typically non-adhesive.

Those of ordinary skill in the art will appreciate that a transdermalpatch is but one example of a device with which inventive nanoparticlecompositions may be administered. To give but a few other examples, adevice may be employed that allows the composition to be applied withoutfirst applying the composition to one's fingers, which may lead toundesirable paralysis of the fingers. Suitable devices include spatulas,swabs, syringes without needles, and adhesive patches. Use of spatulasor swabs, or the like may require the device to be inserted into acontainer containing the composition. Using syringes may be accomplishedby filling the syringe with the composition. The composition may then betopically spread by the spatulas or swabs, or may be expelled from thesyringes onto the person's skin.

In many embodiments of the invention, it may be desirable to limitdelivery of unmodified peptides to only an intended delivery area. Insome embodiments, such limited delivery may be accomplished by utilizingan inventive nanoparticle composition in an application device thatpermits application of the composition to a target site on the skinwithout applying the composition to non-target site areas of the skin.Clearly, a transdermal patch may be utilized to this end. Alternativelyor additionally, if modified peptides are to be applied topically toonly a selected area, other areas may be covered or pre-treated orotherwise protected from exposure.

Exemplification

The following examples are only intended to provide illustrations ofspecific embodiments contemplated by the present invention. The examplesare not intended in any way to be limiting.

EXAMPLE 1 Pentapeptide Nanoparticle Formulation

This example presents peptide nanoparticle compositions comprising ananoemulsion containing a pentapeptide, KTTKS (SEQ ID NO.: 1), that isknown to have biological activity on the skin structures (Katayama etal., supra).

A pentapeptide nanoemulsion preparation was prepared as follows:

-   -   800 mg of soybean oil and 800 mg of Tween 80 were stirred in a        sterile vial for 5 minutes;    -   8.4 ml water with 0.0001 g of the peptide KTTTS (SEQ ID NO.: 1)        was added and stirred for 20 minutes;    -   The sample was homogenized for 1minute;    -   The sample was stirred for 20 minutes; and    -   The sample was microfluidized once at 23,000 psi.

The resulting pentapeptide nanoemulsion was evaluated for particle sizeusing the Malvern Nano S particle sizer capable of sizing particlesbetween about 0.6 nm-6000 nm. The pentapeptide nanoemulsion preparationhad two particle size peaks having an average particle size of 106 nm(Table 2).

TABLE 2 Particle Size Distribution of a Pentapeptide Nanoparticle SizeRange Percent of Particles  10-20 nm 1.3%  21-100 nm 30.2% 101-120 nm10.4% 121-150 nm 22.4% 151-200 nm 19.3% 201-300 nm 14.7% 301-400 nm 1.7%Total 100.0%

EXAMPLE 2 Pentapeptide Nanoparticle Formulation and TransdermalPenetration with Biological Effect

This example presents peptide nanoparticles comprising a nanoemulsioncontaining a pentapeptide, KTTKS (SEQ ID NO.: 1), that is known to havebiological activity on the skin structures (Katayama et al., supra;incorporated herein by reference). This example demonstrates thebiological efficacy on the skin of transdermally applying a peptidenanoparticle, in this case KTTKS (SEQ ID NO.: 1).

Materials and Methods

A pentapeptide nanoemulsion preparation was prepared as follows:

-   -   5.6 g of Labrafac WL 1349 oil and 5.6 g of Tween 80 were stirred        in a sterile beaker for 5 minutes;    -   58.8 g Reagent Grade water was placed in a separate beaker;        0.010 g of the peptide KTTTS (SEQ ID NO.: 1) was added into the        water and stirred for 20 minutes;    -   The contents of the first beaker were added to the contents of        the beaker (i.e., the water and peptide) and then stirred for 20        minutes; and    -   The entire sample was microfluidized once at 23,000 psi.

The resulting pentapeptide nanoemulsion was evaluated for particle sizeusing the Malvern Nano S particle sizer capable of sizing particlesbetween about 0.6 nm- about 6000 nm. The pentapeptide nanoemulsionpreparation had an average particle size of 114.4 nm. Approximately 95%of the particles were below 130 nm in size.

The pentapeptide nanoemulsion was then mixed with an equal volume of askin cream, (Base PCCA Vanishing Cream Light) and then vortexed into auniform cream to yield the “Treatment Cream.”

A “Control Cream” was prepared by the same method as the TreatmentCream, except that no peptide was added in the process.

Ten Swiss Webster mice were purchased that were each approximately 20grams of weight. Upon arrival, all animals were acclimated to theircages for one week (group housed 5 mice per cage per group as definedbelow) and provided with standard cage bedding and Purina 5001 chow.After one week, the following treatment paradigms were applied:

Treatment Paradigms

Group 1 (Control): Each day for eight weeks, 5 mice each had 75 μl ofthe Control Cream applied to their backs with a gloved finger until nocream was visible. The mice had their backs shaved with an electricshaver two days prior to the first treatment and, thereafter, in oneweek intervals.

Group 2 (Treatment): Each day for eight weeks, 5 mice each had 75 μl ofthe Treatment Cream applied to their backs with a gloved finger until nocream was visible. The mice had their backs shaved with an electricshaver two days prior to the first treatment and, thereafter in one weekintervals.

Assessment

The skin from the each mouse's back that was treated with either theControl or Treatment Cream was preserved and then processed withMasson's Trichrome histologic stain. The intensity of the staining wasevaluated at a magnification of 400× using on a histologic scale of 1 to4 for staining intensity: 1=almost no staining, collagen fibrils werevery thin, 2=minimal staining and minimal collagen fibril width,3=moderate staining and moderate fibril width, and 4=intense stainingand wide fibrils.

Results

Histological Assessment

The average histologic score of the skin tissue stained with Masson'sTrichrome stain was 2.33 out of a possible 4 in the Control Group. Bycomparison, the average histologic score of the stained skin of the micein the Treatment Group was 3.67 out of a possible 4. This represents a57% increase in collagen-staining intensity of the Treatment group overthe Control Group. See FIG. 1 for examples of photomicrographs of skintissue specimens from each of the Control and Treatment Groups.

Assessment of Skin Thickness Effects

The thickness of skin thickness is measured using a Skin Layer ThicknessTest to determine the depth (in mm) of each skin layer followingexamination of a histologic cross-section of the mouse back skin thatwas microtomed and placed on a glass slide.

Assessment of Extracellular Matrix Production

Collagen is a major component of the content of the ExtraCellularMatrix. Collagen content is assessed using two separate histologicstains (Picro Sirius Red and Pterocarpus Osun) for collagen in twoseparate tests of mouse back skin that had microtomed and placed on aglass slide.

Collagen content is assessed by using the Western Blot technique todetect the hydroxyproline content of a homogenized preparation of themouse's back skin. Hydroxyproline content is representative of collagencontent.

Conclusion

The result show that, on average, the Treatment Group had statisticallymore collagen than the Control Group by histologic assessment. Thesecontrolled data show that the topical pentapeptide nanoemulsionpreparation had a measurable biological effect on the skin when comparedto a control cream without such a pentapeptide. Prior studies have shownthat the peptide cannot penetrate the intact skin without chemicalmodification (Katayama et al., supra). Therefore, these data show thatthe inventive nanoemulsion formulation enabled penetration of unmodifiedpeptide, resulting in effects to the skin consistent with the knownbiological action of the peptide in increasing collagen production inthe skin and resultant increase in skin thickness.

The results arc expected to show that, on average, the Treatment Groupdoes have statistically thicker skin than the Control Group. The resultsare expected to show that, on average, the Treatment Group does havestatistically more collagen than the Control Group and as measured bythe two histologic stains and Western Blot measurement ofhydroxyproline.

EXAMPLE 3 Skin Thickening and Extra-Cellular-Matrix Stimulator Effectson Mice Through Transdermal Application of a Peptide Nanoparticle:Effect of Varying Concentration of Peptide in the Nanoparticle

This example demonstrates the impact of varying the concentration ofpeptide in the nanoparticle on the biological efficacy on the skin oftransdermally applying a peptide nanoparticle.

Materials and Methods

The experiment described Example 3 is repeated, except that theconcentration of peptide in the Treatment cream is decreased by a factorof ten or increased by a factor of ten.

Results and Conclusion

The results are expected to show that, on average, those mice treatedwith the peptide concentration increased by a factor of ten havestatistically thicker skin those mice treated with the increased peptideconcentration. The results are expected to show that, on average, thosemice treated with the peptide concentration increased by a factor of tenhave statistically more collagen than those with decreased peptideconcentration as measured by the two histologic stains and Western Blotmeasurement of hydroxyproline. In sum, these controlled data areexpected to suggest that biological effect on the skin of the peptidenanoemulsion varies depending on the concentration of peptideincorporated.

Example 4 Administration of Pentapeptide Nanoparticle to Human Subjectsto Reduce Skin Lines

This example demonstrates the biological efficacy on the human skin oftransdermally applying a peptide nanoparticle.

Materials and Methods

A pentapeptide nanoemulsion prepared in accordance with Example 1 or 2is prepared and mixed with an equal volume of a skin cream, (Base PCCAVanishing Cream Light) and then vortexed into a uniform cream to yieldthe “Treatment Cream.”

A “Non-Nano Treatment Cream” is prepared by creating mixing the sameamount of pentapeptide into the same amount of water as Example 1 andthen vortexing with the same amount of skin cream as was used to preparethe Treatment Cream.

A “Control Cream” is prepared by vortexing the same amount of water asExample 1 or 2 and with the same amount of skin cream as was used toprepare the Treatment Cream.

Thirty healthy human subjects with prominent facial lines (such asobserved in people with photo-damaged skin) are enrolled in adouble-blind, placebo-controlled, split-faced study with left-rightrandomization. All subjects are graded with a five point scale by anobserver blinded to treatment status. Score 0 of the scale is normalskin with a score of 5 being severe facial lines and wrinkles (primarilyin the periocular or “crow's feet” area). Cheek skin texture is alsoassessed in terms of pore size (small to large) and smoothness (smoothto rough/pebbly). Subjects are only enrolled if they have a score oninitial examination of 2.5 or greater. The face of the subjects will bephotographed using standardized views and distances and lightingconditions.

Treatment Paradigms

The patient agrees not to use any facial skin care products for 3 weeksexcept for a Control Cream they can use twice daily at 12 hourintervals. After this initial “wash-out” period, each patient is giventwo tubes of cream marked “Right” and “Left” with a unique numericalcode for each tube. They are instructed to use the Right Tube on theright side of the face and the Left Tube on the left side of the facetwice daily at 12 hour intervals. They are instructed to apply a“pea-sized” amount of cream (approximately 0.4 g) to each side of theface. They are also instructed not to use other facial skin careproducts. For 10 of the subjects (the Control Group), the Right Tubecontains the Control Cream and the Left Tube contains the Control Cream.For 10 of the subjects (the Non-Nano Treatment Group), the Right Tubecontains the Control Cream and the Left Tube contains the Non-NanoTreatment Cream. For 10 of the subjects (the Nano Treatment Group), theRight Tube contains the Control Cream and the Left Tube contains theNano Treatment Cream.

Assessment

The subjects are observed and photographed at 4, 8, and 12 weeks afterbeginning the treatment protocols following the wash-out period. Inaddition, an observer blinded to treatment status of the subject as wellas the subject herself scores skin texture with the aforementioned scalefor each of the right and left sides of the face.

Results and Conclusion

The results are expected to show that, on average, the Nano TreatmentGroup has statistically greater differences between the Right and Leftfacial texture scores (showing skin appearance improvement) than thedifferences observed between Right and Left scores for the Control Groupand that the Non-Nano Treatment Group. In sum, these controlled data areexpected to show that the topical pentapeptide nanoemulsion preparationhas a measurable cosmetic effect on the skin when compared to a controlcream without such a pentapeptide and a simple cream (Non-Nano Cream)with the same pentapeptide that was not in a nanoparticle formulation.

Equivalents and Scope

The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. Those of ordinary skill in the art will appreciate that variouschanges and modifications to this description may be made withoutdeparting from the spirit or scope of the present invention, as definedin the following claims.

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention also includes embodiments in which more than one, or all ofthe group members are present in, employed in, or otherwise relevant toa given product or process. Furthermore, it is to be understood that theinvention encompasses all variations, combinations, and permutations inwhich one or more limitations, elements, clauses, descriptive terms,etc., from one or more of the claims or from relevant portions of thedescription is introduced into another claim. For example, any claimthat is dependent on another claim can be modified to include one ormore limitations found in any other claim that is dependent on the samebase claim. Furthermore, where the claims recite a composition, it is tobe understood that methods of using the composition for any of thepurposes disclosed herein are included, and methods of making thecomposition according to any of the methods of making disclosed hereinor other methods known in the art are included, unless otherwiseindicated or unless it would be evident to one of ordinary skill in theart that a contradiction or inconsistency would arise. For example, itis to be understood that any of the compositions of the invention can beused for inhibiting the formation, progression, and/or recurrence ofadhesions at any of the locations, and/or due to any of the causesdiscussed herein or known in the art. It is also to be understood thatany of the compositions made according to the methods for preparingcompositions disclosed herein can be used for inhibiting the formation,progression, and/or recurrence of adhesions at any of the locations,and/or due to any of the causes discussed herein or known in the art. Inaddition, the invention encompasses compositions made according to anyof the methods for preparing compositions disclosed herein.

Where elements are presented as lists, e.g., in Markush group format, itis to be understood that each subgroup of the elements is alsodisclosed, and any element(s) can be removed from the group. It is alsonoted that the term “comprising” is intended to be open and permits theinclusion of additional elements or steps. It should be understood that,in general, where the invention, or aspects of the invention, is/arereferred to as comprising particular elements, features, steps, etc.,certain embodiments of the invention or aspects of the inventionconsist, or consist essentially of, such elements, features, steps, etc.For purposes of simplicity those embodiments have not been specificallyset forth in haec verba herein. Thus for each embodiment of theinvention that comprises one or more elements, features, steps, etc.,the invention also provides embodiments that consist or consistessentially of those elements, features, steps, etc.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and/or the understanding of one of ordinary skill in the art,values that are expressed as ranges can assume any specific value withinthe stated ranges in different embodiments of the invention, to thetenth of the unit of the lower limit of the range, unless the contextclearly dictates otherwise. It is also to be understood that unlessotherwise indicated or otherwise evident from the context and/or theunderstanding of one of ordinary skill in the art, values expressed asranges can assume any subrange within the given range, wherein theendpoints of the subrange are expressed to the same degree of accuracyas the tenth of the unit of the lower limit of the range.

In addition, it is to be understood that any particular embodiment ofthe present invention may be explicitly excluded from any one or more ofthe claims. Any embodiment, element, feature, application, or aspect ofthe compositions and/or methods of the invention (e.g., any peptide, anypeptide modification, any nanoparticle, any nanoemulsion, anysurfactant, any oil, any premix component, any method of preparingnanoemulsions, any method of treatment, etc.), can be excluded from anyone or more claims. For purposes of brevity, all of the embodiments inwhich one or more elements, features, purposes, or aspects is excludedare not set forth explicitly herein.

1. An oil-in-water or water-in-oil dispersion comprising a population ofparticles, wherein the majority of particles have diameters betweenapproximately 10 and approximately 300 nanometers; wherein the particleshave an average particle size ranging between 50 nanometers and about250 nanometers; wherein said particles comprise at least one unmodifiedpeptide of length between 2 and 30 amino acids that has biologicalactivity in the skin, subcutaneous tissue or contiguous muscles; andwherein the at least one unmodified peptide promotes extra-cellularmatrix production, decreases wrinkles, improves wound healing, or treatsexcessive accumulation of extra-cellular matrix. 2.-80. (canceled) 81.The dispersion of claim 1, wherein the majority of particles have arange of diameters between approximately 10 and approximately 250nanometers, or between approximately 10 and approximately 200nanometers, or between approximately 10 and approximately 150nanometers, or between approximately 10 and approximately 120nanometers, or between approximately 10 and approximately 100nanometers, or between approximately 10 and approximately 50 nanometers.82. The dispersion of claim 1, wherein the dispersion is a nanoemulsion.83. The dispersion of claim 82, wherein the nanoemulsion comprises atleast one oil, at least one surfactant, water, and the unmodifiedpeptide.
 84. The dispersion of claim 1, further comprising an oil. 85.The dispersion of claim 84, wherein the oil is selected from the groupconsisting of almond, apricot kernel, avocado, babassu, bergamot, blackcurrent seed, borage, cade, camomile, canola, caraway, carnauba, castor,cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed,emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd,grape seed, hazel nut, hyssop, jojoba, kukui nut, lavandin, lavender,lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoamseed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel,peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran,rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn,sesame, shea butter, silicone, soybean, soy sunflower, tea tree,thistle, tsubaki, vetiver, walnut, and wheat germ oils; butyl stearate;caprylic triglyceride; capric triglyceride; cyclomethicone; diethylsebacate; dimethicone 360; isopropyl myristate; mineral oil;octyldodecanol; oleyl alcohol; silicone oil; short chain triglycerides;medium chain triglycerides; Labrafac WL 1349; long chain triglycerides;saturated oils thereof; and unsaturated oils thereof.
 86. The dispersionof claim 85, wherein the oil is soybean.
 87. The dispersion of claim 85,wherein the oil is a medium chain triglyceride.
 88. The dispersion ofclaim 1, further comprising a surfactant.
 89. The dispersion of claim88, wherein the surfactant is selected from the group consisting ofphosphoglycerides; phosphatidylcholines; dipalmitoyl phosphatidylcholine(DPPC); dioleylphosphatidyl ethanolamine (DOPE);dioleyloxypropyltriethylammonium (DOTMA); dioleoyl-phosphatidylcholine;cholesterol; cholesterol ester; diacylglycerol; diacylglycerolsuccinate;diphosphatidyl glycerol (DPPG); hexanedecanol; fatty alcohols such aspolyethylene glycol (PEG); polyoxyethylene-9-lauryl ether; a surfaceactive fatty acid, such as palmitic acid or oleic acid; fatty acids;fatty acid monoglycerides; fatty acid diglycerides; fatty acid amides;sorbitan trioleate (Span 85) glycocholate; sorbitan monolaurate (Span20); polysorbate 20 (Tween-20); polysorbate 60 (Tween-60); polysorbate65 (Tween-65); polysorbate 80 (Tween-80); polysorbate 85 (Tween-85);polyoxyethylene monostearate; surfactin; a poloxomer; a sorbitan fattyacid ester such as sorbitan trioleate; lecithin; lysolecithin;phosphatidylserine; phosphatidylinositol; sphingomyelin;phosphatidylethanolamine (cephalin); cardiolipin; phosphatidic acid;cerebrosides; dicetylphosphate; dipalmitoylphosphatidylglycerol;stearylamine; dodecylamine; hexadecyl-amine; acetyl palmitate; glycerolricinoleate; hexadecyl sterate; isopropyl myristate; tyloxapol;poly(ethylene glycol)5000-phosphatidylethanolamine; poly(ethyleneglycol)400-mono-stearate; phospholipids; synthetic and/or naturaldetergents having high surfactant properties; deoxycholates;cyclodextrins; chaotropic salts; and ion pairing agents.
 90. Thedispersion of claim 89, wherein the surfactant is a polysorbate.
 91. Thedispersion of claim 1, further comprising an oil and a surfactant. 92.The dispersion of claim 91, wherein the ratio of surfactant to oil isapproximately 0.25:1 by weight; approximately 0.5:1 by weight;approximately 1:1 by weight; approximately 2:1 by weight; orapproximately 3:1 by weight.
 93. The dispersion of claim 1, wherein theat least one unmodified peptide treats hypertrophic scarring, keloids,localized sclerosis, systemic sclerosis, or other conditioncharacterized by excess accumulation of the extra-cellular matrix. 94.The dispersion of claim 1, wherein the at least one unmodified peptidehas a cosmetic effect on the skin.
 95. A method, comprising steps of:administering an amount of an oil-in-water or water-in-oil dispersion toskin of a subject, wherein the dispersion comprises a population ofparticles, wherein the majority of particles have diameters betweenapproximately 10 and approximately 300 nanometers, and wherein theparticles have an average particle size ranging between 50 nanometersand about 250 nanometers; wherein the particles comprises at least oneunmodified peptide of length between 2 and 30 amino acids that hasbiological activity in the skin, subcutaneous tissue or contiguousmuscles, and wherein the at least one modified peptide promotesextra-cellular matrix production, decreases wrinkles, improves woundhealing, or treats excessive accumulation of extra-cellular matrix. 96.The method of claim 95, wherein the subject has at least one symptom offacial wrinkles, and wherein the dispersion is administered to skin ofthe subject in an amount effective to treat, alleviate, ameliorate,relieve, delay onset of, inhibit progression of, reduce severity of, orreduce incidence of, facial wrinkles.
 97. The method of claim 95,wherein the subject has at least one symptom of hypertrophic scarring,keloids, localized sclerosis, systemic sclerosis, or other conditioncharacterized by excess accumulation of the extracellular matrix; andwherein the dispersion is administered to skin of the subject at anamount effective to treat, alleviate, ameliorate, relieve, delay onsetof, inhibit progression of, reduce severity of, or reduce incidence ofthe at least one symptom associated with hypertrophic scarring, keloids,localized sclerosis, systemic sclerosis, or other conditioncharacterized by excess accumulation of the extracellular matrix. 98.The method of claim 95, wherein at least 50% of the unmodified peptidepermeates the skin of the subject.
 99. A method of manufacturing thedispersion of claim 1, the method comprising: providing a premixcomprising an oil, a surfactant, water, and the at least one unmodifiedpeptide; subjecting the premix to high shear force or high pressurehomogenization for a period of time and under conditions which achievethe dispersion of claim 1.